EP0783681B1 - Apparatus and method for carrying out electrochemiluminescence test measurements - Google Patents
Apparatus and method for carrying out electrochemiluminescence test measurements Download PDFInfo
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- EP0783681B1 EP0783681B1 EP94925843A EP94925843A EP0783681B1 EP 0783681 B1 EP0783681 B1 EP 0783681B1 EP 94925843 A EP94925843 A EP 94925843A EP 94925843 A EP94925843 A EP 94925843A EP 0783681 B1 EP0783681 B1 EP 0783681B1
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
- G01N21/69—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence specially adapted for fluids, e.g. molten metal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
- G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
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- G01N2035/0474—Details of actuating means for conveyors or pipettes
- G01N2035/0482—Transmission
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/808—Optical sensing apparatus
Definitions
- This application relates generally to apparatus and methods for detecting the presence of and/or measuring analytes of interest by inducing and detecting electrochemiluminescence in a test sample.
- Chemiluminescent assay techniques have been adopted in which a sample containing an analyte of interest is mixed with a reactant labeled with chemiluminescent label. The reactive mixture is incubated and some portion of the labeled reactant binds to the analyte. After incubation, the bound and unbound fractions of the mixture are separated and the concentration of the label in either or both fractions can be determined by chemiluminescent techniques. The level of chemiluminescence determined in one or both fractions indicates the amount of the analyte of interest in the sample. Electrochemiluminescent (ECL) assay techniques provide improvements over chemiluminescent techniques.
- ECL Electrochemiluminescent
- US-A-5,061,445 describes an apparatus for conducting measurements of electrochemiluminescent (ECL) phenomena which includes a cell unit having an electrode configuration for inducing the emmission of ECL light by the application of a selected voltage wave form to a sample fluid including an ECL moiety.
- the sample fluid is transferred to and from the cell unit by a flow through/tubing system.
- a photomultiplier tube detects the intensity of light emitted by the sample fluid during the ECL measurement process.
- a computer control unit both analyses the detected data and provides digital control signals to the cell unit to generate effective voltage wave forms.
- the digital control signals are supplied to a pulse width modulated digital to analog converted which outputs a ramp voltage wave form having the desired slope.
- Figure 1 an apparatus for use in carrying out electrochemiluminescence test measurements in accordance with one embodiment of the present invention is illustrated in Figure 1 in a generalized block format.
- a flow cell 50 serves to apply electrical energy to an electrochemiluminescent fluid sample in a controlled electrochemical environment which is reproducible, in order to induce electrochemiluminescence thereby thus to enable detection and/or quantitation of an analyte to which an ECL label is bound.
- a light detector 60 is disposed in proximity to the flow cell to receive light 62 emitted by the ECL fluid sample. The light detector 60 produces an electrical signal representing the amount of light received thereby which, after processing, provides a highly accurate measure of the amount of ECL material in the sample.
- the light detector 60 of the Figure 1 embodiment advantageously employs a photomultiplier tube to produce the electrical signal, although other detection devices may be employed.
- the present invention provides temperature effect compensation by carrying out at least one of adjusting a temperature of the electrochemiluminescent fluid sample to a value at least within a predetermined range of temperature values, and adjusting a light output signal representing the light emitted through electrochemiluminescence based on the temperature of the electrochemiluminescent fluid sample to provide a temperature effect adjusted signal.
- the embodiment of Figure 1 serves both to adjust the temperature of the electrochemiluminescent fluid sample as well as to adjust measured values of the light based on the actual temperature of the electrochemiluminescent fluid sample in the flow cell 50.
- Sample fluids supplied to the flow cell 50 are subjected to temperature control to at least bring their temperatures to within a predetermined range of temperatures prior to the conduct of the ECL test in the flow cell.
- a sample fluid heater system 70 and a flow cell temperature control system 80 are provided.
- a fluid handling system 90 serves in general to supply fluids useful for the conduct of ECL tests by the flow cell. Such fluids include cleaning fluids, assay buffers, and air, as well as test sample fluid and calibration sample fluids.
- the fluid handling system 90 receives cleaning fluids, assay buffers and air through respective inlet conduits 100, 101 and 102, and receives the test and calibration sample fluids from tube containers arranged by a user in a sample holder carousel of the fluid handling system 90, described in greater detail bellow.
- control and signal/data processing system 110 Through control lines linking each of these systems and devices therewith.
- the control and signal/data processing system 110 also receives signals in either or both of analog and digital form over signal lines from each of these systems and devices for processing both to assist in exercising its control functions as well as to input signals for processing to produce test result data to be output by the apparatus via a serial I/O port 120 of the control and signal/data processing system 110.
- the control and signal/data processing system 110 is operative to receive programs via the serial I/O port 120 and to store the same for carrying out programmed ECL assays under external control.
- Such received programs are supplied from a personal computer (PC) in communication with the apparatus via the serial I/O port 120.
- PC personal computer
- a user either selects or generates the desired programs with the use of the PC, so that the apparatus of Figure 1 provides substantial versatility in carrying out ECL tests.
- the control and signal/data processing system 110 also carries out temperature compensation of test results based upon temperature data from the flow cell 50, to compensate for any error between the actual sample temperature and a predetermined nominal test temperature.
- the apparatus includes an exterior housing 130 in which all of the elements of Figure 1 embodiment are housed, with the exception of certain components of the fluid handling system 90.
- the fluid handling system 90 includes a sample holder carousel identified in Figure 2 as 140 which serves to releasably support a plurality of sample fluid and calibration fluid holding tubes 142 each at a respective one of a plurality of horizontally spaced sample holder positions arranged in a circular pattern adjacent a periphery of the sample holder carousel 140.
- the carousel 140 serves to rotate about a vertical axis in order to present each of the tube holder positions in a predetermined sequence to a predetermined pipetting position at which the fluid contents of a respective holder tube 142 may be aspirated by a pipetting device indicated generally as 150 in Figure 2 and which is described in greater detail below.
- the carousel 140 includes a rotational base member 160 in a form of a horizontally extending base plate having a generally circular periphery provided with a plurality of gear teeth 162 which may also be seen in Figure 3 .
- the gear teeth 162 provide a means for rotationally driving the base member 160 together with additional elements of the sample holder carousel 140 mounted thereon, as explained below.
- the base member 160 is rotationally mounted on a base support member 170 by means of a bearing 172. Lateral rotational support of the base member 160 is provided by a plurality of ball bearings 180 maintained in a race defined by respective grooves in each of the rotational base member 160 and base support member 170.
- a first horizontally extending circular plate 190 of the carousel 140 is spaced vertically from and affixed to the rotational base member 160 by a plurality of vertically arranged standoffs 200 affixed by screw fasteners to each of the rotational base member 160 and the first circular plate 190.
- a second horizontally extending circular plate 210 arranged parallel to and vertically spaced from.the rotational base member 160 and the first circular plate 190 is movably coupled with the first circular plate 190 by a plurality of shock mount assemblies 220.
- Each of the shock mount assemblies includes a first flexible member 222 affixed to the first circular plate 190 by a screw fastener, a vertically arranged cylindrical stand-off 224 affixed to the first flexible member 222 and extending downwardly therefrom, and a second flexible member 226 affixed to the stand-off 224 at a lower extremity thereof and affixed by a screw fastener to the second circular plate 210.
- the shock mount assemblies 220 permit horizontal movement of the second circular plate 210 with respect to the first circular plate 190 in response to force supplied horizontally to the second circular plate 210, while in the absence of such force, returning the second circular plate 210 to a position generally aligned vertically with the position of the first circular plate 190.
- a sample is mixed with a suitable reagent containing an ECL moiety to bind the ECL moiety to the analyte of interest, if present, or to carry out a competitive binding reaction.
- the result of the reaction is the formation of particulate material to which the ECL moiety is bound.
- the reacted sample in a holder tube 142 is arranged in a respective holding position in the carousel 140.
- the first circular plate 190 and the second circular plate 210 are each provided with a plurality of horizontally spaced interior circular walls 230 and 232, respectively arranged in a circular pattern adjacent the outer horizonal periphery of the each of the first and second plates 190 and 210.
- each of the interior circular walls 232 of the second circular plate 210 is vertically aligned with a respective one of the interior circular walls 230 of the first circular plate.
- each of the holder tubes 142 includes an upper lip 240 defining a mouth of the holder tube 142 and a body portion 242 extending downwardly from the lip 240 to a lower closed end as shown in the illustration of Figure 4 .
- the inner circular walls 230 and 232 of the first and second circular plates 190 and 210 are each dimensioned to receive and engage the body 242 of a respective holder tube 142 at respective positions therealong such that the inner wall 230 of the first circular plate 190 engages the body 242 of the holder tube 142 at a position intermediate the mouth defined by the lip 240 and the position at which the inner circular wall 232 of the plate 210 engages the body 242.
- the fluid samples in the sample holder tubes 142 may contain particulate material including the analyze of interest bound to an ECL label. So that a portion of the sample fluid when aspirated by the pipetting device 150 of Figure 2 will contain a concentration of the particulate material which is representative of the sample overall, it is desired to agitate the sample fluid contained in each of the tubes 142 either prior to or as the sample fluid therein is aspirated by the pipetting device 150. Even where the sample fluid does not contain particulate material, it is desired to agitate the sample fluid either before or during pipetting to ensure a uniform temperature throughout the sample fluid as well as uniform composition thereof.
- a motor system is provided in the carousel 140 which serves to apply horizontal force to the second plate 210 so that the same moves horizontally to agitate the lower portion of each of the sample tubes 142 as each is engaged adjacent a lower extremity thereof by a respective inner circular wall 232 of the plate 210 so that the same moves therewith.
- the force for moving the lower circular plate 210 is provided by a motor 250 held in a motor housing including a lower member 252 affixed by a screw fastener to a third circular plate 260 which, in turn, is affixed to an upper member 270 of the motor housing.
- the first circular plate 190 and an upwardly extending handle 280 are both affixed to the upper member 270 by a screw fastener.
- the handle 280 is provided with an inner wall 282 of generally frustoconical shape at a bottom surface of which an electrical connector 284 is fastened and coupled electrically with the motor 250 for providing power thereto.
- the plug 284 is connected with a power cord 290 coupled with a source of power of the apparatus within the exterior housing 130 to controllably energize the motor 250.
- the configuration of the plug 284 permits the plug 284 to rotate with respect to the cord 290.
- the motor 250 has a motor shaft 300 coupled with a half-round counterweight 310.
- the shaft 300 is also coupled with a second shaft 320 having a shaft axis offset from a shaft axis of the motor shaft 300 and journaled for rotation in a bearing 330 affixed to the second circular plate 210.
- the offset shaft 320 will likewise rotate while describing a circular translatory motion about the axis of the motor shaft 300. Since the offset shaft 320 is journaled for rotation in the bearing 320 affixed to the second plate 210, the second plate 210 will move with the axis of the offset shaft 320 so that the plate 210 likewise moves with respect to the first plate 190 against force exerted by the shock mount assemblies 220 tending to return the second plate 210 to its rest position with respect to the first plate 190.
- each of the holder tubes 140 which are engaged by the inner circular walls 232 of the second plate 210 will likewise move with the plate 210 in order to agitate the fluid contents of each of the tubes 142.
- the half-round counterweight 310 balances the system to avoid excessive vibration thereof.
- the third circular plate 260 is provided with a plurality of inner circular walls 340 each of which is aligned with a respective one of the inner circular walls 230 of the first circular plate 190 and is dimensioned to receive the body of a respective one of the tubes 142.
- the carousel 140 also includes a cylindrical wall 350 which extends entirely about the lower member 252 of the motor housing and is spaced relatively close to the positions of the holder tubes 142 to the interior thereof.
- An outer surface of the cylindrical wall 350 has a flat black finish to provide desired optical properties as explained hereinbelow.
- Figure 6 illustrates a motor assembly 360 which serves to rotationally drive the rotational base member 160 of the carousel 140, the motor assembly 360 being mounted to a base plate 362 which is independent of the carousel 140. That is, the carousel 140 is supported by its base member 170 independently of the base plate 362.
- the motor assembly 360 also includes a gear 370, shown partially broken away, rotatably mounted on a bearing 376 mounted on a idler arm 380 which, in turn, is mounted on the base plate 362.
- gear 370 shown partially broken away, rotatably mounted on a bearing 376 mounted on a idler arm 380 which, in turn, is mounted on the base plate 362.
- the motor assembly 360 also includes a motor 390 mounted on a motor arm 392 which, in turn, is rotationally mounted on a shaft 396.
- the motor 390 has a rotational shaft 400 on which a motor gear 402 is mounted.
- the motor arm 392 is biased by a spring (not shown for purpose of simplicity and clarity) to bring the motor gear 402 into mesh with the gear 370.
- the gear 370 and the gear teeth 162 of the base member 160 may be replaced by a suitable belt drive or frictional drive.
- the holder mechanism includes a base plate 410 on which a lever 420 is mounted for rotation in a horizontal plane about a pivot 422.
- a latch 430 is pivotally mounted on a slidable carrier 440 for rotation about a pivot 442 in a vertical plane.
- the latch 430 has a latching end 446 opposite a second end thereof on which cam surface 450 is formed.
- the lever 420 has a corresponding cam surface which engages the cam surface 450 of the lever 420 so that when the lever 420 is rotated toward the latching end 446 of the latch 430, the latching end 446 is raised to release the rotational base member 160 of the carousel 140, thus to permit rotation thereof.
- the latching end 446 of the latch 430 is urged downwardly by a coil spring 460 positioned between the base plate 410 and a portion of the latch 430 on a side of the pivot 422 opposite the latching end 446.
- the lever 420 when the lever 420 is rotated away from the latch 430, the latching end 446 thereof is forced downwardly by the coil spring 460 to engage and retain the rotational base member 160 of the sample holder carousel to prevent rotation thereof.
- the position of the slidable carrier 440, and thus the position of the latch 430 with respect to the base plate 410 is adjustable by means of a set screw 464.
- the rotation of the lever 420 is actuated by a linear actuator (not shown for purpose of simplicity and clarity) under the control of the system 110 as appropriate to either maintain the carousel in a stationary state or permit it to rotate to present a new tube holder position to the pipetting device 150.
- a homing notch 470 is formed in the first circular plate 190 at a predetermined rotational position thereof.
- the apparatus is provided with an optical interrupter 472 which produces an output signal indicating to the system 110 of Figure 1 when the homing notch 470 has been brought into alignment therewith. At that point, the apparatus has determined the absolute rotational position of the carousel 140.
- the motor 390 which preferably is a stepper motor, is actuated to rotate the carousel 140 by a predetermined rotational amount to bring a first holder tube position of the carousel 140 into alignment with the pipetting device 150.
- the apparatus includes a tube presence detector system 480 as illustrated in Figure 9 .
- the detector system 480 detects the presence of a tube 142 at the pipetting position through the detection of light reflected therefrom. This avoids the need to employ mechanical devices, such as switches, for this purpose, thus avoiding the disadvantage of mechanical wear and the eventual need to replace such devices.
- the tube presence detector system 480 includes an infra-red emitting diode (IRED) 490 positioned adjacent the pipetting device 150 to project light 496 toward any tube 142 which may be present at the pipetting position.
- the system 480 also includes a photodiode 500 vertically aligned with the IRED 490 and disposed to receive light 502 reflected from any tube 142 at that position.
- the IRED 490 is driven to emit the light 496 in a pulsed fashion in response to a pulsed drive current supplied by a voltage controlled current amplifier 510.
- An oscillator 520 produces a corresponding pulsed output signal having a 5% duty cycle and a period of 1.6 milliseconds.
- the voltage controlled current amplifier produces the driving current supplied thereby to the IRED 490 which likewise has a 5% duty cycle and 1.6 millisecond period.
- the IRED can be driven at a high current level to emit light pulses of relatively high intensity thus to assist in enabling the system 480 to distinguish reflected pulses from background light levels.
- the wall 350 ( Figure 4 ) has a flat black finish, in the absence of a tube 142 at the pipetting position of the carousel 140, only a relatively small amount of the light will be reflected back towards the photodiode 500 from the wall 350.
- An output of the photodiode 500 is coupled with the input of a preamplifier 530 having a band pass characteristic centered on the frequency of the pulses produced by the oscillator 520, which thus serves to assist in rejecting both DC outputs from the photodiode as well as 60 and 120 Hz components from ambient lighting in order to reduce stray light sensitivity of the system 480.
- An output of the preamplifier 530 is coupled with a first input of a comparator 540 having a second input supplied with a selectable threshold level, as described in greater detail hereinbelow and providing a binary level output.
- the selectable threshold level is chosen so that, in the absence of a tube 142 at the pipetting position, the signal output by the preamplifier 530 will result in a first state of the output from the comparator 540, while when a tube 142 is present at the pipetting position, the comparator 540 outputs a pulsed binary level signal having the same frequency and duty cycle as the output of the preamplifier 530.
- the output of the comparator 540 is supplied to the D input of a D-type flip-flop 550 which has a clock input terminal coupled with the output of the oscillator 520.
- the flip-flop 550 is caused to latch the output of the comparator 540 at the end of the IRED 490 drive on-state thus to synchronize sampling of the signal produced by the light-receiving portion of the system 480 with the pulsed light output by the transmitting portion thereof.
- the output of the flip-flop 550 is supplied to the control and signal/data processing system 110 thus to provide the system 110 with the ability to determine whether a tube is present at the pipetting position so that the pipetting device 150 may be actuated to aspirate a sample therefrom as appropriate.
- the system 110 supplies the selectable threshold level in digital form to the input of a digital-to-analog converter 560 which latches this value and outputs the same in analog form both to the second input of the comparator 540 as well as to a voltage controlled gain input terminal of the voltage controlled current amplifier 510.
- the foregoing arrangement permits the system 110 to control the sensitivity of the tube presence detector system 480 through a relatively wide dynamic operating range. That is, since both the gain of the amplifier 510 as well as the threshold level of the comparator 540 are controlled by the same signal supplied by the DAC 560, the sensitivity of the system is proportional to the square of the DAC output so that the system's dynamic range is extended as compared with the dynamic range of the DAC 560 output.
- the system 110 By providing the system 110 with the ability to control the sensitivity of the system 480, it is possible for the system 480 to be adjusted to reliably detect the presence of the tubes 142 even though tubes of different colors and materials may be employed which may reflect different amounts of light and even though variations in the positions and dispositions of the tubes 142 may be encountered.
- the system 480 is easily calibrated to compensate for IRED's 490 and photodiodes 500 having different characteristics, as well as to compensate for the effects of aging in these components.
- tubes 142 are placed in a number of specified positions in the carousel 140 and the carousel is advanced both to positions where tubes are known to be present as well as positions where it is known that no tubes are present.
- the system 110 supplies a digital ramp signal to the DAC 560 and stores the value thereof at which the flip-flop 550 toggles, this value being referred to as a "calibration threshold".
- a detection threshold for use in detecting the presence of a tube 142 in normal operation is derived by taking the average of the two calibration thresholds constituting (1) the lowest calibration threshold obtained for the positions at which a tube is present, and (2) the highest calibration threshold obtained for the positions in which a tube is not present. Subsequently, in normal operation the system 110 writes the detection threshold into the DAC 560 for use by the system 480 for detecting tube presence in normal operation.
- a closed container 580 containing a cleaning fluid is coupled by the line 100 of the fluid handling system 90 to a further heater system 590, while a further closed container 600 containing assay buffer is coupled by the inlet line 101 to the heater system 590.
- the heater system 590 serves to raise the temperatures of the cleaning fluid and assay buffer supplied from the containers 580 and 600 to within a predetermined temperature range in order to assist in maintaining a desired temperature of the flow cell 50 as well as the remainder of the fluid handling system 90 preceding the flow cell 50 to assist in achieving temperature control of sample fluids subjected ECL tests in the flow cell 50.
- the operation of the heater systems 70 and 590 will be explaining hereinbelow in greater detail.
- the cleaning fluid is supplied via a line 610 to a first inlet of a manifold valve 620, while a further line 630 conducts the assay buffer to a second inlet of the manifold valve 620.
- a third line 640 open to the air is connected with a third inlet of the manifold valve 620.
- the manifold valve 620 is solenoid actuated and is operative in response to control signals received thereby to select one of the cleaning fluid, assay buffer and air to be supplied to an outlet thereof coupled with a manifold outlet line 650.
- the pipetting device 150 includes a valve block 660 shown in Figure 10 and illustrated in cross-section in Figure 11 .
- An inlet 670 of the valve block 660 is coupled with the manifold outlet line 650 to receive the fluid supplied by the manifold valve 620.
- the pipetting device 150 includes a probe 680 slidably mounted with respect to the valve block 660 so that the same may be lowered into a respective holder tube 142 to remove liquid therefrom or, in the alternative, raised to a retracted position as shown in Figure 11 .
- the probe 680 is affixed to a coupling 690 shown in partial cross-section as having a fitting to receive a pipetting device outlet line 700 ( Figure 10 ).
- the coupling 690 is mounted on a slidable block 710 slidably mounted on a shaft 720 acting as a linear bearing to guide the block 710 and attached probe 680 as the same is raised and lowered.
- the block 710 is fitted with a threaded aperture (not shown for purposes of simplicity and clarity) mated with the threads of a lead screw 730.
- the lead screw 730 is rotatably coupled with a stepper motor (not shown for purposes of simplicity and clarity) which is controllably operable to rotate the lead screw 730 in either of two selectable directions to controllably raise or lower the block 710 and the attached pipetting probe 680.
- the lead screw 730 and shaft 720 are supported by a base 732.
- the pipetting probe 680 is slidably received in a fitting 740 mated with the value block 660 to provide a fluid tight seal therebetween.
- a poppet mechanism (not shown for purposes of simplicity and clarity) coupled with a spring mounted seal 750 engages the seal 750 in a lower opening 760 of the valve block 660 to form a fluid tight seal therewith.
- fluids may be conveyed via the manifold outlet line through the valve block inlet 670 to the probe 680 in order to convey cleaning fluid, assay buffer and/or air to the portion of the fluid handling system 90 downstream of the valve block 660 through the outlet line 700.
- cleaning fluid admitted to the valve block through the inlet 670 serves to clean the lower portion of the probe 680.
- the poppet mechanism When the slidable block 710 is lowered by appropriately rotating the lead screw 730, the poppet mechanism is actuated to withdraw the seal 750 from the lower opening 760 to permit the probe 680 to descend from the valve block 660 into a respective one of the holder tubes 142 to aspirate fluid therefrom.
- a pair of O-ring seals 766 and 768 are forced against a shoulder of the valve block 660 by a washer 770 affixed to the probe 680 to form a fluid tight seal therewith.
- the outlet line 700 is coupled with a T-junction 780 having a first outlet coupled with a line 790 through which fluids are conveyed to the heater block 570 of the sample fluid heater system 70 ( Figure 1 ) to be heated thereby in order to bring the fluids conveyed via the line 790 substantially to a predetermined temperature for the conduct of an ECL measurement by the flow cell 50 which receives the heated fluid from an outlet line 802 of the heater block 570.
- Fluid received at an inlet of the flow cell 50 from the outlet line 802 is ultimately conveyed via an outlet thereof to a first inlet of a bypass valve 810, a second inlet of the bypass valve 810 being coupled with a second outlet of the T-junction 780.
- the bypass valve 810 is a solenoid valve operative to couple either the outlet of the flow cell 50 or the second outlet of the T-junction 780 to an outlet line 820 of the bypass valve 810.
- the outlet line 820 is coupled with an inlet of a peristaltic pump 830 which serves to controllably draw fluids through the fluid handling system 90, heater block 570 and flow cell 50.
- An outlet of the peristaltic pump 830 is coupled with a waste fluid container 840 for disposal of used fluids.
- the flow cell 50 is mounted within an environmentally controlled housing 850, illustrated in Figure 12 .
- the housing 850 has a photomultiplier tube (PMT) 860 of the light detector system 60 mounted on an upper surface of the housing 850 and positioned to receive light produced through electrochemiluminescence in the flow cell 50 mounted beneath the PMT 860 within the housing 850.
- the housing 850 is sealed against stray light on all sides as well as at all openings, for example, where the PMT 860 is mated to the housing 850.
- the housing 850 is also insulated against heat conduction therethrough by an insulating cover 870 shown partially broken away for ease of illustration.
- the temperature within the housing 850 is controlled by means of the flow cell temperature control system 80 of Figure 1 which serves to apply heat to the exterior of the housing 850 to maintain its interior temperature substantially at a predetermined value by means of foil heaters 880 adhesively affixed to three lateral sides of the housing 850 as well as to a bottom surface thereof. Further details of the flow cell temperature control system will be explained hereinbelow.
- the heater block 570 is mounted on an exterior lateral surface of the housing 850 and is fabricated of a metal, such as brass, providing good heat conductivity.
- the line 790 through which sample fluids, as well as cleaning fluids, assay buffers and air pass on their way to the flow cell 50 are conducted through the heater block 800 to adjust their temperatures to within at least a predetermined range of temperatures to permit the conduct of ECL tests on the sample fluids in a reproducible manner.
- the fluids emitted from the heater block 570 are conveyed via the line 802 through the housing 850 and, as shown in Figure 10 , to the flow cell 50.
- a temperature sensor 890 is affixed to the heater block 800 to produce a signal representing the temperature thereof.
- two power transistors 900 are affixed to the heater block in order to controllably apply heat thereto for maintaining the temperature of the heater block at a desired level.
- the sample fluid heater system 70 is implemented as a proportional/integral temperature controller in order to provide close correspondence between a desired temperature of the heater block 570 and the actual temperature thereof.
- a desired or set temperature of the heater block 570 is written to a DAC 910 by the control and signal/data processing system 110 so that the set temperature value in analog form is output by the DAC 910 to a first input of a difference amplifier 920.
- a second input of the difference amplifier 920 is provided with the output of the temperature sensor 890 and the difference amplifier 920 serves to produce an error voltage representing the difference between the set or desired temperature of the block 570 and the actual temperature thereof as sensed by the temperature sensor 890.
- the error voltage output by the difference amplifier 920 is supplied to the input of an analog-to-digital converter 930, as well as to a first input of a summing amplifier 940.
- An output of the summing amplifier 940 is supplied to the input of a driver 950 which serves to provide a controlled heating current to the power transistors 900 for controllably heating the block 800.
- the loop represented by the temperature sensor 890, difference amplifier 920, summing amplifier 940 and driver 950 represents a proportional controller loop, that is, a control loop in which the heating current is proportional to the difference between the set temperature and the measured temperature.
- the system 70 In the operation of a practical proportional control loop having a realistic gain (that is, a gain which is sufficiently limited to avoid instability and consequent oscillation), there typically is a steady state error between the measured value (here the actual temperature of the block 570) and the desired value (that is, the set temperature). Consequently, the system 70 also employs an integral controller loop which is implemented by the analog-to-digital converter 930, the control and signal/data processing system 110 acting as an integrator 960 as illustrated in Figure 13 , together with a digital-to-analog converter 970 which serves to convert the output of the integrator 960 to analog form and supply the same to a second input of the summing amplifier 940.
- the integral control loop After each data acquisition from the analog-to-digital converter 930, adds the converted error voltage to an integral term which is stored by the system 110. This value is then scaled by an appropriate gain factor and written to the DAC 970 to be output in analog form to the second input of the summing amplifier 940.
- the output of the DAC 970 serves to substantially eliminate the steady state error associated with the proportional control loop.
- the integral value is modified to accommodate design limitations of the system. That is, when the error voltage is sufficiently large that the proportional control system alone will drive the power transistors at maximum power, the integral term is set by the system 110 to zero. In addition, if the integral term becomes sufficiently large that it likewise will drive the power transistors at full power, the amount of the integral is prevented from increasing so that it does not accumulate past a point where it can have any further effect on the temperature of the heater block 570.
- FIG. 14 a block diagram of the flow cell temperature control system 80 is illustrated therein. While the flow cell temperature control system 80 of Figure 1 may be implemented in the manner illustrated in Figure 13 for the sample fluid heater system 70, the system as illustrated in Figure 14 , is implemented entirely by the control and signal/data processing system 110. That is, the flow cell temperature control system 80 includes a temperature sensor 960 mounted on the flow cell 50 within the housing 850 and is coupled with an analog-to-digital converter 970 which digitizes the output of the temperature sensor 960 and provides the same to the system 110 acting as a control loop processor 980.
- a temperature sensor 960 mounted on the flow cell 50 within the housing 850 and is coupled with an analog-to-digital converter 970 which digitizes the output of the temperature sensor 960 and provides the same to the system 110 acting as a control loop processor 980.
- the control loop processor 980 carries out both the proportional and integral processing functions as performed by the system 70 of Figure 13 (described hereinabove) and outputs a digital value to the input of a digital-to-analog converter (DAC) 990 representing a drive current to be applied to the foil heaters 880 adhesively affixed to the exterior of the housing 850 of Figure 12 .
- the DAC 990 converts the drive value to analog form and supplies the same to the input of a driver 1000 which serves to apply a corresponding heating current to the foil heaters 880.
- the system 80 of Figure 14 can be eliminated and the foil heaters 880 driven instead by the driver 950 of the system 70 ( Figure 13 ).
- cooling elements may likewise be used to establish a predetermined test temperature.
- Such cooling elements include, for example thermoelectric coolers and Peltier coolers.
- a further alternative is to subject the apparatus to a temperature controlled medium, either liquid or gas (such as air).
- the flow cell 50 is affixed to the housing 850 and spaced slightly below the upper surface thereof so that light emitted through electrochemiluminescence within the flow cell 50 propagates towards the PMT 860 to be converted thereby to an electrical signal representing an amount of light received thereby.
- the flow cell 50 includes an arm 1010 pivotally mounted thereto, the arm having a permanent magnet 1020 affixed thereto so that the magnet 1020 may be pivoted either to a position in the vicinity of a working electrode of the flow cell 50 for use in collecting magnetic particles with bound ECL labels pursuant to a magnetic particle assay, or away from the flow cell 50, for example, when electrochemiluminescence of the ECL labels is induced in order to avoid interference with the operation of the PMT 860.
- the arm 1020 is coupled through a coil spring 1030 with an arm of a solenoid operated linear actuator 1040.
- a motor driven fan 1050 is mounted within the housing 850 and runs continuously to circulate air within the housing 850 to maintain a substantially uniform temperature throughout its interior.
- a circuit board 1060 is mounted on the flow cell 50.
- the circuit board 1060 includes circuitry for coupling the working electrode as well as counter electrodes and a reference electrode included in the flow cell 50 with the system 110 for the purpose of applying voltage and current to the counter and working electrodes and to measure such voltages and currents as well as a voltage level on the reference electrode.
- Circuit board 1060 also includes a reference LED 1070 which may be energized selectively to emit a controlled amount of light toward the PMT 860 to enable calibration thereof.
- the flow cell 50 includes a main housing 1080 fabricated of a durable, transparent and chemically inert material which is easy to machine or injection mold to the configuration illustrated in Figures 16-18 .
- Suitable materials for the housing 1080 include acrylic and polymethyl methacrylate.
- the main housing 1080 has a first lower surface 1090 ( Figure 17 ) through which a fluid inlet defined by a threaded coupling 1100 and contiguous conduit 1110 are formed in the main housing 1080. As seen in Figure 17 , the conduit 1110 extends from the threaded coupling 1100 to an upper surface 1120 of the main housing 1080.
- a fluid outlet is also formed in the main housing 1080 and includes a threaded coupling 1130 extending upwardly from a second lower surface 1140 of the main housing 1080 to a further conduit 1150 which extends therefrom to the upper surface 1120.
- An ECL test chamber or container 1174 is formed between the upper surface 1120 of the main housing 1180 and a lower surface of a transparent block 1160 affixed above the upper surface 1120 and separated therefrom by a gasket 1170 which defines lateral walls of the chamber 1174.
- the gasket 1170 forms a fluid tight seal between the block 1160 and the main housing 1080, the block 1160 and gasket 1170 being held to the main housing 1180 by a plurality of fasteners 1180 ( Figure 16 ).
- the chamber 1174 thus defined by the main housing 1080, the block 1160 and the gasket 1170 communicates with the conduit 1110 adjacent a first lateral side of the chamber 1174 and with the conduit 1150 at a second lateral side of the chamber 1174 opposite the first lateral side. Accordingly, fluids introduced through the fluid inlet defined by the coupling 1100 and conduit 1110 flow through the chamber 1174 from right to left as viewed in Figure 17 and are emitted therefrom through the fluid outlet formed by the conduit 1150 and threaded coupling 1130, so that the fluid inlet, the chamber and the fluid outlet define a fluid flow path through the flow cell 50.
- a working electrode 1182 is arranged in a shallow groove formed in the upper surface 1120 of the main housing 1180 and has a longitudinal axis arranged generally transverse to a longitudinal axis of the chamber 1174 extending from the first lateral side thereof to its second lateral side and is positioned laterally centrally thereof between the conduits 1110 and 1150.
- the working electrode 1182 is held within the shallow groove in the top surface 1120 of the main housing 1080 by means of a first retainer block 1230 held against the surface 1120 by a pair of fasteners and serving to maintain a first electrical lead (not shown for purposes of simplicity and clarity) in conductive contact with the working electrode for coupling the same with the circuit board 1060.
- a first counter electrode 1190 is arranged to extend along a bottom surface of the block 1160 forming an upper surface of the chamber 1174 from a position approximately opposite a first lateral side of the working electrode 1182 toward the first lateral side of the chamber 1174 adjacent the conduit 1110 and therebeyond between the gasket 1170 and the block 1160 and is forward upwardly at a right angle to extend along a first lateral side of the block 1160.
- the first counter electrode 1190 is held against the first lateral side of the block 1160 by a second retainer block 1210 fastened by a pair of fasteners to the block 1160 and which also serves to securely connect an electrical lead (not shown for purposes of simplicity and clarity) to the first counter electrode 1190 to couple the same with the circuit board 1060 ( Figure 15 ).
- a second counter electrode 1200 extends along the bottom surface of the block 1160 from a position approximately opposite a second lateral side of the working electrode 1182 outwardly along the top wall of the chamber 1174 formed by the bottom wall of the block 1160 toward the conduit 1150 and therebeyond between the block 1160 and the gasket 1170 to a second lateral edge of the block 1160 where the second counter electrode is formed at a right angle to extend upwardly therealong.
- a third retainer block 1220 retains the second counter electrode against the second lateral side of the block 1160 by means of a further pair of fasteners and serves to securely couple a further electrical lead (not shown for purposes of simplicity and clarity) to the second counter electrode 1200 for coupling the same to the circuit board 1060.
- Materials suitable for the working electrode 1182 and the counter electrodes 1190 and 1200 include platinum and gold.
- the counter electrodes 1190 and 1200 are arranged on a wall (that is, the bottom surface of the block 1160) of the chamber opposite a second wall thereof (that is, the upper surface 1120 of the main housing 1080) on which the working electrode 1182 is arranged.
- Conventional flow cells place the working and counter electrodes on the same wall of the chamber in which ECL is induced so that the emitted light can pass through an opposite transparent wall of the chamber to be detected by a PMT.
- certain electrode materials are prone to flake off as fluid flows by, thus tending to form a conductive bridge which shorts out the counter and working electrodes, thereby rendering the flow cell unusable until the conductive bridge has been removed by cleaning.
- the flow cell 50 as shown particularly in Figure 17 substantially alleviates this problem by positioning a counter electrode on a wall of the ECL chamber opposite a wall thereof on which the working electrode is arranged but positioned so that the surface of the working electrode does not oppose the counter electrode, but rather is arranged opposite a wall which is made of transparent material. Consequently, any material which may flake off either a counter electrode or the working electrode will not tend to form a conductive bridge between the counter and working electrodes, while at the same time light emitted by ECL labels adjacent the surface of the working electrode can be transmitted through the transparent wall to be detected.
- a further advantage provided by the flow cell 50 of Figures 16-18 is provided by the arrangement of counter electrodes 1190 and 1200 on opposite sides of the working electrode 1182 which serves to minimize variations in the flow of electric current between the counter electrodes 1190 and 1200, on the one hand, and the working electrode 1182, on the other, which may be caused, for example, by variations in fluid flow or composition within the ECL chamber 1174.
- a magnet 1020 is mounted on an arm 1010 which is pivotally connected with the flow cell 50.
- the arm 1010 and magnet 1020 are illustrated therein in an upper position in which the magnet 1020 is brought in close proximity to the working electrode, being separated therefrom only by a relatively narrow wall 1240 of the main housing 1080.
- the magnet 1020 serves to accumulate magnetic particles bound to ECL labels adjacent a surface of the working electrode exposed to fluids in the ECL chamber 1174 in carrying out magnetic particle assays. Since it is desirable to move the magnet 1020 downwardly away from the PMT 860 when ECL measurements are carried out, the arm 1010 is mounted to the main housing 1080 by a pivot pin 1250.
- a reference electrode 1260 includes, for example, a wire immersed in an ionic solution permanently retained by an outer glass housing capped at an outer end by a glass frit which permits ionic communication between the ionic fluid within the glass housing and fluids which may come in contact with the glass frit.
- Conventional flow cell structures bring the glass frit of the reference electrode directly in contact with fluids within the fluid flow path, so that ionic exchange takes place therewith and the chemical composition of the ionic fluid within the glass housing of the reference electrode gradually changes so that the electrical characteristics of the reference electrode change or drift disadvantageously over time.
- the flow cell 50 of Figures 16-18 substantially alleviates this problem by interposing a further ionic fluid between the flow path and the ionic fluid within the reference electrode.
- the second ionic fluid is retained within a chamber 1274 formed by the main housing 1080 and a lateral block 1270 held to the main housing by a plurality of fasteners and sealed thereagainst by a further gasket 1280.
- the block 1270 may be made, for example, of the same material as the main housing 1080.
- the reference electrode 1260 is inserted into the chamber formed between the block 1270 and the main housing 1080 to bring its glass frit into contact with an ionic conductive medium therein.
- a glass or ceramic frit 1290 is positioned in an aperture within the main housing 1080 joining the conduit 1150 and the chamber 1274 and is retained therein by a plug 1300 which presses against an 0-ring seal 1310 to seal the outer periphery of the frit 1290 against invasion of fluids from the conduit 1150 or loss of ionic conductive media within the chamber 1274 to the conduit 1150.
- a refill aperture is formed in the upper surface 1120 of the main housing 1080 extending to the chamber 1274 and is sealed by a removable plug 1320 which permits an ionic media within the chamber 1274 to be replaced.
- a suitable ionic conductive medium for filling the chamber 1274 is a gel including sodium chloride and agarose having a concentration selected to render the gel solid at room temperature, but liquefiable at 80°C so that the same may be poured into the chamber 1274 through the aperture in the upper surface 1120 of the main housing 1080.
- the gel also contains phenolphthalein providing an indicator to detect leaks across the frit 1290. In particular, the phenolphthalein turns the gel pink when cleaning fluid from the conduit 1150 comes in contact with the gel due to a change in pH of the gel brought about by the cleaning fluid.
- the temperature sensor 960 of the temperature control system of Figure 14 is mounted on the flow cell 50 within the housing 850 of Figure 15 . As shown in Figure 16 , the temperature sensor 960 is mounted on a side wall of the main housing 1080.
- FIGS 19A through 19C provide a block diagram of the control and signal/data processing system 110 of the Figure 1 embodiment.
- a central processing unit 1330 including a microprocessor, microcomputer or the like, is bidirectionally coupled with a RS 232 serial interface 1340 coupled with serial input/output port 120 for data communication.
- the CPU 1330 is also coupled bidirectionally with a memory 1350 including a RAM as well as nonvolatile storage, for example, provided by flash memory circuits.
- the CPU 1330 is operative to communicate with an external source of assay control programs through the interface 1340 to receive and store such programs in the memory 1350.
- the external programming source may be, for example, a personal computer in which a user inputs programs through a keyboard, disk drive, or other input device.
- the control and signal/data processing unit 110 is operative to receive and store a plurality of assay control programs, as well as to run such programs simultaneously to provide a multitasking capability which promotes efficient use of the apparatus.
- by permitting a user to create and run multiple programs it is possible for the user to design and test relatively small portions of a more complex assay which greatly facilitates assay development.
- the CPU 1330 is also bidirectionally coupled with a timer processor unit (TPU) 1360 in the form of a programmable timer device operative to generate and read clock signals.
- TPU timer processor unit
- the TPU 1360 is employed to generate stepper motor drive signals as well as to convert voltage-to-frequency converted values to a digital form which may be processed by the CPU 1330.
- the CPU 1330 is also bidirectionally coupled with an input/output unit 1370 which provides a digital communication capability between the CPU 1330 and various peripheral detection and drive circuits, as explained in greater detail below.
- the input/output unit 1370 is bidirectionally coupled with a digital input/output circuit 1380 which serves both as a digital multiplexer and dimultiplexer for digital signals provided from the input/output unit 1370 and various peripheral digital circuits, as well as for buffering various digital signals to be communicated to and from the peripheral circuits.
- the digital input/output unit 1380 is coupled with a stepper motor control circuit 1390, which is also coupled with the TPU 1360 to receive a stepper motor step pulse signal.
- the stepper motor control circuit 1390 serves to buffer direction and enable signals generated by the CPU 1330 and supplied via the digital input/output circuit 1380 for use in generating appropriate control signals to drive a selected one of the stepper motors of the apparatus selectably in high or low power mode and in normal or reverse direction.
- the stepper motor circuit 1390 includes separate latches to store direction and enable signals for each of the carousel rotating motor 390 ( Figure 6 ), the peristaltic pump 830 ( Figure 10 ), the linear actuator 1040 ( Figure 15 ) and the probe up/down drive motor described in connection with Figure 11 .
- the stepper motor control circuit is coupled with an optical interface and drive circuit 1400 to provide the various control signals for controlling the various stepper motors.
- the optical interface and drive circuit 1400 serves to decouple voltage spikes generated by the stepper motors from the remainder of the system 110 as well as to generate the necessary drive signals.
- the circuit 1380 is also coupled with a valve driver circuit 1410 which serves to latch control signals for controlling the states of the solenoids in the manifold 620 as well as the state of the solenoid controlling the bypass valve 810, both as illustrated and described in connection with Figure 10 .
- the circuit 1410 likewise includes suitable driver circuits for driving the valve solenoids in accordance with the latched control signals.
- the manifold 620 includes three valves, each controlled by a respective solenoid for either communicating or blocking access from a respective inlet of the manifold 620 to the outlet line 650 thereof.
- the circuit 1380 also receives the output of the D-type flip-flop 550 of the tube presence detection system of Figure 9 and latches the same to be provided to the CPU 1330 for detecting the presence of a holder tube 142 at the pipetting position.
- the circuit 1380 has a plurality of inputs 1430 for receiving temperature detection signals from the various temperature control systems described hereinabove.
- the circuit 1380 has an input coupled with an optical switch interface circuit 1440 which, in turn, receives detection signals from the optical interrupter 472 of Figure 9 (for detecting the homing position of the carousel 140), a home sensor for the probe 680 of Figure 11 , a home sensor for the pump 830 of Figure 10 and a sensor providing a signal indicating whether the exterior housing 130 has been opened, in order to provide the system with the ability to shut down high voltage supplies in that event.
- the interface circuit 1440 conditions the detection signals from the various optical interruptors and latches the same for providing appropriate outputs to the circuit 1380 for provision to the CPU 1330 for control purposes.
- the digital input/output circuit 1380 has a serial output coupled with a serial input of a control digital-to-analog converter 1450 which serves to latch digital values provided by the circuit 1380 and convert the same to analog form for carrying out various control functions described in greater detail hereinbelow. More particularly, the control DAC 1450 latches a threshold level signal for tube presence detection and converts the same to analog form which it supplies over an output 1460 to the comparator 540 of Figure 9 (so that the control DAC 1450 implements the function of the DAC 560 as shown in Figure 9 ).
- control DAC 1450 latches a digital value representing a drive voltage for the agitation motor 250 and outputs the same in analog form to an agitation motor drive circuit 1470 which, in turn, provides a driving current to the motor 250.
- the control DAC 1450 also latches set temperatures for each of the temperature control systems 70 and 80 of Figure 1 as well as the system 590 of Figure 10 and outputs the same in analog form over a plurality of output lines indicated as 1480 in Figure 19B .
- control DAC 1450 latches a digital value received from circuit 1380 representing a high voltage level to be applied to the PMT 860 and converts the same to analog form which it supplies to a PMT high voltage power supply 1490 for controlling the high voltage applied thereby to the PMT 860.
- the digital input/output circuit 1380 outputs digital values representing reference LED drive level and waveform generation parameters to a digital-to-analog converter (DAC) 1500 having a plurality of addressable latches for storing these values to be supplied respectively to a reference LED drive circuit 1510 for supplying an appropriate drive level to the reference LED 1070 of Figure 15 and to a waveform generator 1520 which serves to generate waveforms appropriate for driving the electrodes of the flow cell 50 for carrying out ECL measurements, as well as for cleaning and conditioning the electrodes.
- the DAC 1500 also receives a reference voltage level from a voltage reference circuit 1530.
- the waveform generator 1520 selectably generates either a ramp voltage waveform having a slope endpoint specified by the value supplied by the DAC 1500 or else a specified, constant output voltage.
- the waveforms thus produced by the waveform generator 1520 are supplied to an input of a potentiostat 1540.
- the potentiostat 1540 is coupled with each of the reference, counter and working electrodes and serves to apply the waveform received from the waveform generator 1520 so that the voltage level appearing at the reference electrode corresponds with the voltage output by the waveform generator 1520. Since the reference electrode does not conduct current, it will be seen with reference to Figure 17 that the reference electrode will have a voltage level which is essentially the same as the voltage level on the counter electrode 1200.
- the counter electrode 1200 is coupled with the counter electrode 1190 on the circuit board 1060, so that the voltage level at the counter electrode 1190 is the same as that at the counter electrode 1200. Moreover, until current begins to flow between the counter electrodes and the working electrode 1182, the voltage level at the working electrode 1182 will be essentially the same as that on the counter electrodes and the reference electrode. However, once current begins to flow from the counter electrodes to the working electrode in response to a drive voltage applied between the counter and working electrodes by the potentiostat 1540, the voltage level at the surface of the working electrode falls below that of the counter and reference electrodes in proportion to the amount of current flowing between the counter and working electrodes.
- the potentiostat 1540 produces a current sensing voltage representing current flowing between the counter and working electrodes, as well as values representing electrode.voltage levels and supplies these signal in analog form to a first input of a multiplexer and voltage-to-frequency converter 1550 having a plurality of inputs at which it receives respective analog voltages to be multiplexed and converted to signals in the frequency domain which, in turn, it supplies to the digital input/output circuit 1380 and timer processor unit 1360 for conversion to a form suitable for processing by the CPU 1330.
- the circuit 1550 also receives the output of the waveform generator 1520, the reference voltage from the circuit 1530 and a temperature detection signal from the temperature sensor 960 ( Figure 16 ) for multiplexing and conversion in the same manner as the signals received from the potentiostat 1540.
- a luminometer 1560 receives the output of the PMT 860 and provides both a low gain output on an output terminal 1562 and a high gain output on an output line 1564 each of which is coupled with a respective input of the multiplexer and voltage-to-frequency converter 1550. The provision of low and high gain outputs from the luminometer 1560 provides a wide dynamic range of operation for the apparatus.
- the circuit 1550 has an input coupled to receive a ground level reference input.
- Figure 20 provides a diagram illustrating the functional relationships among basic program elements of the software which controls the operation of the CPU 1330 of Figure 19A .
- the system 110 employs a multitasking operating system 1570 on which a supervisor program 1580 runs for managing the overall operation of the system 110 and, therefore, the apparatus overall.
- the software also includes binary sequences 1590 each of which may be called by a higher level command to carry out a relatively specific, predefined task. Also included are a plurality of sequence engines 1600 each of which operates independently of the other sequence engines and acts as an interpreter for higher level commands, calling the binary sequences 1590 as appropriate to execute these commands.
- a number of device drivers 1610 execute commands which control instrument hardware, such as valves, stepper motors, and the like by outputting appropriate digital control signals via the input/output unit 1370 of Figure 19A .
- the device drivers 1610 also control the storage of newly received assay control programs in non-volatile memory included within the memory block 1350 of Figure 19A .
- a data link controller 1620 manages the data communications via the RS232 interface 1340, including activities such as packeting, routing and error checking.
- the supervisor program serves to initialize the system, including setting up the device drivers 1610, initializing hardware and starting the data link controller task.
- the supervisor program starts a sequence engine 1600 in response to a command received by the serial input/output port 120 and then assumes a background status, awaiting an event requiring its intervention.
- Such events include, for example, a system error, a request for a system reset, and a failure of the data link, in which case some or all of the system may require initialization by the supervisor program.
- the supervisor has initiated a plurality of sequence engines 1600 to run simultaneously, it serves to keep track of the system conditions overall and responds to any conflict between instructions carried out by different sequence engines in order to resolve the same.
- Table I provides a summary of commands available to a user for programming the operation of the embodiment of Figure 1 , which may be entered via the serial input/output port 120 individually or in the form of an assay control program which is stored and selectably run by the control and signal/data processing system 110.
- the Acquire command requires an argument specifying the type of data to be captured while the Heater command requires an argument specifying the particular heating system 70, 80 or 590 for which the temperature is to be set.
- the Acquire command also starts or stops a data capture activity, as specified by the argument.
- a temperature compensation task is carried out.
- the memory 1350 stores a table of values which specify the amount of an adjustment which must be made in a given ECL luminosity reading depending on the deviation of the actual sample fluid temperature from a nominal testing temperature.
- the memory means stores data representing temperature dependence of light produced through electrochemiluminescence.
- the temperature measured by the temperature sensor 960 mounted on flow cell 50 is employed to access the appropriate compensation data for this purpose.
- Figures 21A and 21B provide a flow chart of a main processing loop of the exemplary assay.
- the apparatus is initialized and assay parameters are specified, as indicated in step 1630. That is, the apparatus is reset by the Ireset command, followed by the specification of the parameters, the number of holder tubes 142 to be sampled in the course of the assay and the carrying out of a system cleaning subroutine Clean-line 1640.
- Figures 22A and 22B provide a flow chart of the Clean-line subroutine.
- the Clean-line subroutine proceeds to actuate the cleaning fluid solenoid valve of the manifold to switch cleaning fluid to the outlet line 650 of the manifold 620, as indicated in step 1650 with the use of the Valve command summarized in Table I.
- the peristaltic pump is started by means of the Pump command and in a subsequent step 1670 a constant voltage level V c of the waveform generator is produced by means of the Volt command to set the voltage of the flow cell 50 substantially at the level V c in order to draw a cleaning fluid through the flow cell 50 at a predetermined cleaning voltage.
- the bypass valve is turned on and off a predetermined number (N) of times by means of the Valve command in order to expel foreign material which may have become trapped in the T junction 780 ( Figure 10 ).
- the manifold air inlet is turned on and off N times which serves to inject slugs of airs into the system (while the pump remains on) to mechanically dislodge particulate matter which is then carried away by the cleaning fluid.
- the assay buffer valve of the manifold is turned on (while the cleaning solution valve is turned off) to introduce assay buffer into the system.
- the clean line subroutine is concluded by stopping the pump in the step 1730.
- the program Upon return to the main loop as illustrated in Figures 21A and 21B , the program begins executing a program loop including steps 1740 through 1790 repeatedly until all of the sample tubes have been read.
- the carousel is first moved to the position of the next tube by means of the Carousel command in step 1740. Thereafter a Startube subroutine 1750 is called.
- the Startube subroutine serves to draw a sample fluid from a holder tube 142 at the pipetting position.
- the pump is brought to a home position in order to permit a precise amount of the sample to be withdrawn from the tube.
- the vortexing motor is turned on by the Vortex command and thereafter the assay buffer valve of the manifold is opened by the Valve command in a step 1820.
- the pump is turned on to draw the assay buffer through the flow cell while a succession of voltage ramps is applied in the step 1840 to condition the working electrode by bringing it into a reproducible electrochemical condition by either removing or forming an oxide layer at its working surface. Thereafter the voltage is maintained at a preset value in order to apply a predetermined constant potential to the working electrode, so that the working electrode is conditioned to ensure reproducible test results.
- the vortexing motor is turned off (step 1850), the pump is turned off and returned to its home position (step 1860) and the assay buffer valve is closed (step 1870), in preparation to aspirate the sample from the holder tube.
- the probe 680 is lowered into the holder tube pursuant to the Probe command in step 1880, and the magnet is brought to its up position in a step 1890 pursuant to the Magnet command in order to attract magnetic particles with bound ECL labels to the surface of the working electrode when the sample fluid enters the flow cell 50.
- the sample is drawn into the probe in a succession of steps 1900, 1910 and 1920 pursuant to which the pump is turned on and maintained in the on state for a predetermined period of time determined by means of the Idel command in step 1910 at the end of which the pump is turned off in a step 1920.
- the pump is turned off, a precisely measured amount of the sample has been drawn into the probe 680 which is then withdrawn from the sample tube by means of the Probe command, as indicated in a step 1930 and processing returns to the main processing loop of Figures 21A and 21B .
- the program Upon return to the main loop, the program calls a Trans subroutine in a step 1760 during which the sample fluid is drawn through the flow cell 50 at a controlled rate for the purpose of accumulating the magnetic particles in the fluid adjacent the working electrode in a controlled manner to ensure reproducibility of the test results.
- the assay buffer valve of the manifold is turned on to supply assay buffer to the outlet line 650 thereof in a step 1940.
- a subsequent step 1950 the pump is turned on to draw assay buffer into the system for a predetermined period of time and at a controlled rate so that the sample fluid which precedes the assay buffer in the fluid transfer system is controllably drawn through the flow cell 50, as mentioned above, followed by assay buffer to remove sample particles which have not been captured by the magnet at the surface of the working electrode.
- the cleaning fluid valve of the manifold is turned on (and the assay buffer valve turned off) for a predetermined period of time to introduce cleaning fluid into the system, although not yet into the flow cell 50.
- a Measure subroutine (step 1770) is called for carrying out the ECL measurement.
- a dark current level of the PMT 860 is first obtained in a step 2000 (which actually represents three commands, namely, Acquire dark current level on, followed by Idel for a predetermined time period, and then Acquire dark current level off).
- a further Acquire command is executed to commence ECL data capture in a step 2010, whereupon a suitable sequence of ramp voltage waveforms are applied to the flow cell 50, as indicated in a step 2020, in order to controllably induce electrochemiluminescence by the sample fluid in the flow cell 50.
- the Acquire command is again executed to end ECL data capture, as indicated in step 2030.
- the flow cell voltage is set at zero (step 2040)
- an excitation voltage is applied to the reference LED by executing the Lumref command (step 2050) and an Acquire sequence (step 2060) is carried out to capture PMT readings to provide a reference for evaluating the operating state of the PMT.
- the reference LED is turned off (step 2070) and the program returns once again to the main loop.
- the Clean-line subroutine (step 1780) is again carried out and, in the subsequent step 1790, it is determined whether all of the sample tubes have been read. If not, the program returns to the step 1740 to begin a further measurement sequence to measure the sample contents of the next holder tube.
- step 1790 proceeds to a step 2080 in which the various apparatus devices are turned off, followed by a step 2090 in which the various valves of the apparatus are closed, after which the control and signal/data processing system 110 is brought to a stand-by condition in step 2100 to complete the assay.
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Abstract
Description
- This application relates generally to apparatus and methods for detecting the presence of and/or measuring analytes of interest by inducing and detecting electrochemiluminescence in a test sample.
- Numerous methods and systems have been developed for the detection and quantitation of analytes of interest in biochemical and biological substances. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of substantial value to researchers and clinicians.
- Chemiluminescent assay techniques have been adopted in which a sample containing an analyte of interest is mixed with a reactant labeled with chemiluminescent label. The reactive mixture is incubated and some portion of the labeled reactant binds to the analyte. After incubation, the bound and unbound fractions of the mixture are separated and the concentration of the label in either or both fractions can be determined by chemiluminescent techniques. The level of chemiluminescence determined in one or both fractions indicates the amount of the analyte of interest in the sample. Electrochemiluminescent (ECL) assay techniques provide improvements over chemiluminescent techniques. They provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. In ECL techniques the incubated sample is exposed to a voltammetric working electrode in order to trigger luminescence. In the proper chemical environment, such electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner. Light produced by an electrochemiluminescent label is measured to provide an indication of the presence of the analyte or to measure the same.
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US-A-5,061,445 describes an apparatus for conducting measurements of electrochemiluminescent (ECL) phenomena which includes a cell unit having an electrode configuration for inducing the emmission of ECL light by the application of a selected voltage wave form to a sample fluid including an ECL moiety. The sample fluid is transferred to and from the cell unit by a flow through/tubing system. A photomultiplier tube detects the intensity of light emitted by the sample fluid during the ECL measurement process. A computer control unit both analyses the detected data and provides digital control signals to the cell unit to generate effective voltage wave forms. The digital control signals are supplied to a pulse width modulated digital to analog converted which outputs a ramp voltage wave form having the desired slope. - While ECL techniques have been developed for use in the laboratory, there is a need for practical ECL instrument capable of carrying out multiple assays in an efficient and reproducible manner.
- It is an object of the present invention to provide improved apparatus for carrying out electrochemiluminescence test measurements.
- It is another object of the invention to provide such an apparatus which is versatile and easy to use.
- It is a further object of the invention to provide an electrochemiluminescence test apparatus which affords reproducible and accurate ECL test results.
- It is still another object of the present invention to provide an electrochemiluminescence test apparatus which operates efficiently.
- In accordance with one aspect of the present invention, there is provided an apparatus as defined in Claim 1 of the present specification. Preferred features of the apparatus are defined in the dependent claims.
- According to another aspect of the present invention, there is provided a method for carrying out electrochemiluminescence test measurements as defined in the last claim of the present specification.
- The above, and other objects, features and advantages of the invention will be apparent in the following detailed description of certain illustrative embodiments thereof which is to be read in connection with the accompanying drawings forming a part hereof, and wherein corresponding parts and components are identified by the same reference numerals in the several views of the drawings.
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Figure 1 is a block diagram of an embodiment of an automated electrochemiluminescence test apparatus in accordance with the present invention; -
Figure 2 is a perspective, exterior view of the electrochemiluminescence test apparatus of theFigure 1 embodiment; -
Figure 3 is a partially broken away, top plan view of a sample holder carousel of the embodiment ofFigures 1 and2 ; -
Figure 4 is a cross-sectional view of the sample holder carousel ofFigure 3 taken along the lines 4-4 therein; -
Figure 5 is a cross-sectional view of the sample holder carousel ofFigures 3 and4 taken along the lines 5-5 inFigure 4 ; -
Figure 6 is a side elevational view of a motor system for rotationally driving the sample holder carousel ofFigures 3-5 ; -
Figure 7 is a top plan view of a mechanism for releasably securing the sample holder carousel ofFigures 3-5 against rotation; -
Figure 8 is a cross-sectional view taken along the lines 8-8 inFigure 7 ; -
Figure 9 is a block diagram of a sample tube detector system for detecting the presence of a sample tube in a predetermined sample tube support position of the carousel ofFigures 3-5 ; -
Figure 10 is a schematic diagram of a fluid handling system of theFigure 1 embodiment functionally connected with a sample fluid heater system and flow cell thereof for supplying fluids thereto and removing fluids therefrom; -
Figure 11 is a partially cross-sectional, elevational view of a sample pipetting device in combination with a valve block forming a part of the fluid handling system ofFigure 10 ; -
Figure 12 is a partially broken away, perspective view of a flow cell housing and certain structurally related components of theFigure 1 embodiment; -
Figure 13 is a block diagram of a sample fluid heater system of theFigure 1 embodiment; -
Figure 14 is a block diagram of a temperature control system for the flow cell housing ofFigure 12 ; -
Figure 15 is a cross-sectional view taken along the lines 15-15 inFigure 12 ; -
Figure 16 is a perspective view of the flow cell of theFigure 1 embodiment; -
Figure 17 is a cross-sectional view taken along the lines 17-17 inFigure 16 ; -
Figure 18 is a cross-sectional view taken along the lines 18-18 inFigure 17 ; -
Figures 19A through 19C together provide a block diagram of a control and signal/data processing system of theFigure 1 embodiment; -
Figure 20 is a functional block diagram of software used for controlling the operation of a central processor unit of the control and signal/data processing system ofFigures 19A through 19C ; -
Figures 21A and21B together provide a flow chart of a main processing loop of an exemplary assay control program input to the system ofFigures 19A through 19C ; -
Figures 22A and22B together provide a flow chart of a system cleaning sub-routine called by the main processing loop ofFigures 21A and21B ; -
Figures 23A and 23B together provide a flow chart of a tube sampling sub-routine called by the main loop ofFigures 21A and21B ; -
Figure 24 is a flow chart of a flow cell fluid transfer sub-routine called by the main loop ofFigures 21A and21B ; and -
Figure 25 is a flow chart of an ECL measurement sub-routine called by the main loop ofFigures 21A and21B . - With reference now to the drawings, an apparatus for use in carrying out electrochemiluminescence test measurements in accordance with one embodiment of the present invention is illustrated in
Figure 1 in a generalized block format. - A
flow cell 50 serves to apply electrical energy to an electrochemiluminescent fluid sample in a controlled electrochemical environment which is reproducible, in order to induce electrochemiluminescence thereby thus to enable detection and/or quantitation of an analyte to which an ECL label is bound. Alight detector 60 is disposed in proximity to the flow cell to receivelight 62 emitted by the ECL fluid sample. Thelight detector 60 produces an electrical signal representing the amount of light received thereby which, after processing, provides a highly accurate measure of the amount of ECL material in the sample. Thelight detector 60 of theFigure 1 embodiment advantageously employs a photomultiplier tube to produce the electrical signal, although other detection devices may be employed. - Heretofore, the substantial sensitivity of the electrochemiluminescence process to the temperature of a sample under test, as well as the non-linear character of such temperature dependance, have not been appreciated. In one aspect, the present invention provides temperature effect compensation by carrying out at least one of adjusting a temperature of the electrochemiluminescent fluid sample to a value at least within a predetermined range of temperature values, and adjusting a light output signal representing the light emitted through electrochemiluminescence based on the temperature of the electrochemiluminescent fluid sample to provide a temperature effect adjusted signal. The embodiment of
Figure 1 serves both to adjust the temperature of the electrochemiluminescent fluid sample as well as to adjust measured values of the light based on the actual temperature of the electrochemiluminescent fluid sample in theflow cell 50. Sample fluids supplied to the flow cell 50 (as well as cleaning and conditioning fluids, as explained in greater detail hereinbelow), are subjected to temperature control to at least bring their temperatures to within a predetermined range of temperatures prior to the conduct of the ECL test in the flow cell. For this purpose, both a samplefluid heater system 70 and a flow celltemperature control system 80 are provided. - A
fluid handling system 90 serves in general to supply fluids useful for the conduct of ECL tests by the flow cell. Such fluids include cleaning fluids, assay buffers, and air, as well as test sample fluid and calibration sample fluids. Thefluid handling system 90 receives cleaning fluids, assay buffers and air throughrespective inlet conduits fluid handling system 90, described in greater detail bellow. - Overall control of the operations carried out by the
flow cell 50, thelight detector 60, thetemperature control systems fluid handling system 90, is exercised by a control and signal/data processing system 110 through control lines linking each of these systems and devices therewith. The control and signal/data processing system 110 also receives signals in either or both of analog and digital form over signal lines from each of these systems and devices for processing both to assist in exercising its control functions as well as to input signals for processing to produce test result data to be output by the apparatus via a serial I/O port 120 of the control and signal/data processing system 110. In addition, the control and signal/data processing system 110 is operative to receive programs via the serial I/O port 120 and to store the same for carrying out programmed ECL assays under external control. Typically, such received programs are supplied from a personal computer (PC) in communication with the apparatus via the serial I/O port 120. A user either selects or generates the desired programs with the use of the PC, so that the apparatus ofFigure 1 provides substantial versatility in carrying out ECL tests. - The control and signal/
data processing system 110 also carries out temperature compensation of test results based upon temperature data from theflow cell 50, to compensate for any error between the actual sample temperature and a predetermined nominal test temperature. - Referring also to
Figure 2 , an exterior perspective view of the apparatus ofFigure 1 is there provided wherein certain elements of thefluid handling system 90 ofFigure 1 are visible. As shown inFigure 2 , the apparatus includes anexterior housing 130 in which all of the elements ofFigure 1 embodiment are housed, with the exception of certain components of thefluid handling system 90. As mentioned hereinabove, thefluid handling system 90 includes a sample holder carousel identified inFigure 2 as 140 which serves to releasably support a plurality of sample fluid and calibrationfluid holding tubes 142 each at a respective one of a plurality of horizontally spaced sample holder positions arranged in a circular pattern adjacent a periphery of thesample holder carousel 140. Thecarousel 140 serves to rotate about a vertical axis in order to present each of the tube holder positions in a predetermined sequence to a predetermined pipetting position at which the fluid contents of arespective holder tube 142 may be aspirated by a pipetting device indicated generally as 150 inFigure 2 and which is described in greater detail below. - The
sample holder carousel 140 is now described in detail with reference toFigures 3-5 . The structural relationship of the various elements of thecarousel 140 is seen in the cross-sectional view ofFigure 4 . As shown inFigure 4 thecarousel 140 includes arotational base member 160 in a form of a horizontally extending base plate having a generally circular periphery provided with a plurality ofgear teeth 162 which may also be seen inFigure 3 . Thegear teeth 162 provide a means for rotationally driving thebase member 160 together with additional elements of thesample holder carousel 140 mounted thereon, as explained below. Thebase member 160 is rotationally mounted on a base support member 170 by means of abearing 172. Lateral rotational support of thebase member 160 is provided by a plurality ofball bearings 180 maintained in a race defined by respective grooves in each of therotational base member 160 and base support member 170. - A first horizontally extending
circular plate 190 of thecarousel 140 is spaced vertically from and affixed to therotational base member 160 by a plurality of vertically arrangedstandoffs 200 affixed by screw fasteners to each of therotational base member 160 and the firstcircular plate 190. A second horizontally extendingcircular plate 210 arranged parallel to and vertically spaced from.therotational base member 160 and the firstcircular plate 190 is movably coupled with the firstcircular plate 190 by a plurality ofshock mount assemblies 220. Each of the shock mount assemblies includes a firstflexible member 222 affixed to the firstcircular plate 190 by a screw fastener, a vertically arranged cylindrical stand-off 224 affixed to the firstflexible member 222 and extending downwardly therefrom, and a secondflexible member 226 affixed to the stand-off 224 at a lower extremity thereof and affixed by a screw fastener to the secondcircular plate 210. Theshock mount assemblies 220 permit horizontal movement of the secondcircular plate 210 with respect to the firstcircular plate 190 in response to force supplied horizontally to the secondcircular plate 210, while in the absence of such force, returning the secondcircular plate 210 to a position generally aligned vertically with the position of the firstcircular plate 190. - In exemplary ECL assay method, a sample is mixed with a suitable reagent containing an ECL moiety to bind the ECL moiety to the analyte of interest, if present, or to carry out a competitive binding reaction. In certain assays, the result of the reaction is the formation of particulate material to which the ECL moiety is bound. However, this is not an exhaustive explanation of all types of ECL assay methods.
- After the binding reaction has taken place, the reacted sample in a
holder tube 142 is arranged in a respective holding position in thecarousel 140. For this purpose, the firstcircular plate 190 and the secondcircular plate 210 are each provided with a plurality of horizontally spaced interiorcircular walls second plates circular plate 210 is at rest (that is, the absence of the horizontal force applied thereto), each of the interiorcircular walls 232 of the secondcircular plate 210 is vertically aligned with a respective one of the interiorcircular walls 230 of the first circular plate. - As will be seen with reference to
Figure 4 , each of theholder tubes 142 includes anupper lip 240 defining a mouth of theholder tube 142 and abody portion 242 extending downwardly from thelip 240 to a lower closed end as shown in the illustration ofFigure 4 . The innercircular walls circular plates body 242 of arespective holder tube 142 at respective positions therealong such that theinner wall 230 of the firstcircular plate 190 engages thebody 242 of theholder tube 142 at a position intermediate the mouth defined by thelip 240 and the position at which the innercircular wall 232 of theplate 210 engages thebody 242. - After the binding reaction the fluid samples in the
sample holder tubes 142 may contain particulate material including the analyze of interest bound to an ECL label. So that a portion of the sample fluid when aspirated by thepipetting device 150 ofFigure 2 will contain a concentration of the particulate material which is representative of the sample overall, it is desired to agitate the sample fluid contained in each of thetubes 142 either prior to or as the sample fluid therein is aspirated by thepipetting device 150. Even where the sample fluid does not contain particulate material, it is desired to agitate the sample fluid either before or during pipetting to ensure a uniform temperature throughout the sample fluid as well as uniform composition thereof. - For this purpose, a motor system is provided in the
carousel 140 which serves to apply horizontal force to thesecond plate 210 so that the same moves horizontally to agitate the lower portion of each of thesample tubes 142 as each is engaged adjacent a lower extremity thereof by a respective innercircular wall 232 of theplate 210 so that the same moves therewith. It will be appreciated that, since thebody 242 of thetube 142 is engaged adjacent thelip 240 by the first circular plate which remains stationary with respect to therotational base member 160, thelip 240 remains substantially in a stationary position as the lower portion of thebody 242 is thus agitated. Accordingly, it is possible to reliably introduce thepipetting device 150 into thetube 142 and withdraw a portion of the fluid sample as the tube is agitated. Moreover, since only a portion of thecarousel 140 is subjected to agitation (principally the second circular plate 210), this function of the apparatus is relatively efficiently carried out. - The force for moving the lower
circular plate 210 is provided by amotor 250 held in a motor housing including alower member 252 affixed by a screw fastener to a thirdcircular plate 260 which, in turn, is affixed to anupper member 270 of the motor housing. The firstcircular plate 190 and an upwardly extendinghandle 280 are both affixed to theupper member 270 by a screw fastener. Thehandle 280 is provided with aninner wall 282 of generally frustoconical shape at a bottom surface of which anelectrical connector 284 is fastened and coupled electrically with themotor 250 for providing power thereto. - As shown in
Figure 2 , theplug 284 is connected with apower cord 290 coupled with a source of power of the apparatus within theexterior housing 130 to controllably energize themotor 250. The configuration of theplug 284 permits theplug 284 to rotate with respect to thecord 290. Themotor 250 has amotor shaft 300 coupled with a half-round counterweight 310. With reference also toFigure 5 theshaft 300 is also coupled with asecond shaft 320 having a shaft axis offset from a shaft axis of themotor shaft 300 and journaled for rotation in abearing 330 affixed to the secondcircular plate 210. Accordingly, as the shaft of themotor 250 rotates, the offsetshaft 320 will likewise rotate while describing a circular translatory motion about the axis of themotor shaft 300. Since the offsetshaft 320 is journaled for rotation in thebearing 320 affixed to thesecond plate 210, thesecond plate 210 will move with the axis of the offsetshaft 320 so that theplate 210 likewise moves with respect to thefirst plate 190 against force exerted by theshock mount assemblies 220 tending to return thesecond plate 210 to its rest position with respect to thefirst plate 190. Consequently, the lower portion of each of theholder tubes 140 which are engaged by the innercircular walls 232 of thesecond plate 210 will likewise move with theplate 210 in order to agitate the fluid contents of each of thetubes 142. The half-round counterweight 310 balances the system to avoid excessive vibration thereof. - The third
circular plate 260 is provided with a plurality of innercircular walls 340 each of which is aligned with a respective one of the innercircular walls 230 of the firstcircular plate 190 and is dimensioned to receive the body of a respective one of thetubes 142. Thecarousel 140 also includes acylindrical wall 350 which extends entirely about thelower member 252 of the motor housing and is spaced relatively close to the positions of theholder tubes 142 to the interior thereof. An outer surface of thecylindrical wall 350 has a flat black finish to provide desired optical properties as explained hereinbelow. -
Figure 6 illustrates amotor assembly 360 which serves to rotationally drive therotational base member 160 of thecarousel 140, themotor assembly 360 being mounted to abase plate 362 which is independent of thecarousel 140. That is, thecarousel 140 is supported by its base member 170 independently of thebase plate 362. - The
motor assembly 360 also includes agear 370, shown partially broken away, rotatably mounted on abearing 376 mounted on aidler arm 380 which, in turn, is mounted on thebase plate 362. When thecarousel 140 is brought into position adjacent the apparatus ofFigure 1 , the teeth ofgear 370 mesh with thegear teeth 162 of therotational base member 160 of thecarousel 140. - The
motor assembly 360 also includes amotor 390 mounted on amotor arm 392 which, in turn, is rotationally mounted on ashaft 396. Themotor 390 has arotational shaft 400 on which amotor gear 402 is mounted. Themotor arm 392 is biased by a spring (not shown for purpose of simplicity and clarity) to bring themotor gear 402 into mesh with thegear 370. In other embodiments, thegear 370 and thegear teeth 162 of the base member 160 (Figures 4 and5 ) may be replaced by a suitable belt drive or frictional drive. - Referring now to
Figures 7 and 8 , a mechanism as illustrated therein for releasably retaining therotational base member 160, and therefore the holder tubes carried thereby, against rotational movement while fluids are aspirated from the tubes by thepipetting device 150. The holder mechanism includes abase plate 410 on which alever 420 is mounted for rotation in a horizontal plane about apivot 422. Alatch 430 is pivotally mounted on aslidable carrier 440 for rotation about apivot 442 in a vertical plane. Thelatch 430 has alatching end 446 opposite a second end thereof on whichcam surface 450 is formed. Thelever 420 has a corresponding cam surface which engages thecam surface 450 of thelever 420 so that when thelever 420 is rotated toward thelatching end 446 of thelatch 430, the latchingend 446 is raised to release therotational base member 160 of thecarousel 140, thus to permit rotation thereof. Thelatching end 446 of thelatch 430 is urged downwardly by acoil spring 460 positioned between thebase plate 410 and a portion of thelatch 430 on a side of thepivot 422 opposite thelatching end 446. Consequently, when thelever 420 is rotated away from thelatch 430, the latchingend 446 thereof is forced downwardly by thecoil spring 460 to engage and retain therotational base member 160 of the sample holder carousel to prevent rotation thereof. The position of theslidable carrier 440, and thus the position of thelatch 430 with respect to thebase plate 410 is adjustable by means of aset screw 464. The rotation of thelever 420 is actuated by a linear actuator (not shown for purpose of simplicity and clarity) under the control of thesystem 110 as appropriate to either maintain the carousel in a stationary state or permit it to rotate to present a new tube holder position to thepipetting device 150. - Referring again to
Figure 3 , in order to determine the rotational position of thecarousel 140, a homingnotch 470 is formed in the firstcircular plate 190 at a predetermined rotational position thereof. Referring also toFigure 9 , the apparatus is provided with anoptical interrupter 472 which produces an output signal indicating to thesystem 110 ofFigure 1 when the homingnotch 470 has been brought into alignment therewith. At that point, the apparatus has determined the absolute rotational position of thecarousel 140. - Thereafter, the
motor 390, which preferably is a stepper motor, is actuated to rotate thecarousel 140 by a predetermined rotational amount to bring a first holder tube position of thecarousel 140 into alignment with thepipetting device 150. - Since, depending on the assay being conducted, it is uncertain that a
sample holder tube 142 will be present at each of the holder tube positions of thecarousel 140, when the carousel is rotated to each holder tube position in succession at the pipetting position, it is necessary to determine whether aholder tube 142 is then present at the pipetting position. In order to make this determination, the apparatus includes a tubepresence detector system 480 as illustrated inFigure 9 . Thedetector system 480 detects the presence of atube 142 at the pipetting position through the detection of light reflected therefrom. This avoids the need to employ mechanical devices, such as switches, for this purpose, thus avoiding the disadvantage of mechanical wear and the eventual need to replace such devices. - More particularly, the tube
presence detector system 480 includes an infra-red emitting diode (IRED) 490 positioned adjacent thepipetting device 150 to project light 496 toward anytube 142 which may be present at the pipetting position. Thesystem 480 also includes aphotodiode 500 vertically aligned with theIRED 490 and disposed to receive light 502 reflected from anytube 142 at that position. - The
IRED 490 is driven to emit the light 496 in a pulsed fashion in response to a pulsed drive current supplied by a voltage controlledcurrent amplifier 510. Anoscillator 520 produces a corresponding pulsed output signal having a 5% duty cycle and a period of 1.6 milliseconds. Accordingly, the voltage controlled current amplifier produces the driving current supplied thereby to theIRED 490 which likewise has a 5% duty cycle and 1.6 millisecond period. By limiting the duty cycle of the current pulse produced by theamplifier 510 to substantially 5% (or such lesser amount as may be practical), the IRED can be driven at a high current level to emit light pulses of relatively high intensity thus to assist in enabling thesystem 480 to distinguish reflected pulses from background light levels. In addition, since the wall 350 (Figure 4 ) has a flat black finish, in the absence of atube 142 at the pipetting position of thecarousel 140, only a relatively small amount of the light will be reflected back towards thephotodiode 500 from thewall 350. - An output of the
photodiode 500 is coupled with the input of apreamplifier 530 having a band pass characteristic centered on the frequency of the pulses produced by theoscillator 520, which thus serves to assist in rejecting both DC outputs from the photodiode as well as 60 and 120 Hz components from ambient lighting in order to reduce stray light sensitivity of thesystem 480. An output of thepreamplifier 530 is coupled with a first input of acomparator 540 having a second input supplied with a selectable threshold level, as described in greater detail hereinbelow and providing a binary level output. The selectable threshold level is chosen so that, in the absence of atube 142 at the pipetting position, the signal output by thepreamplifier 530 will result in a first state of the output from thecomparator 540, while when atube 142 is present at the pipetting position, thecomparator 540 outputs a pulsed binary level signal having the same frequency and duty cycle as the output of thepreamplifier 530. The output of thecomparator 540 is supplied to the D input of a D-type flip-flop 550 which has a clock input terminal coupled with the output of theoscillator 520. Accordingly, the flip-flop 550 is caused to latch the output of thecomparator 540 at the end of theIRED 490 drive on-state thus to synchronize sampling of the signal produced by the light-receiving portion of thesystem 480 with the pulsed light output by the transmitting portion thereof. The output of the flip-flop 550 is supplied to the control and signal/data processing system 110 thus to provide thesystem 110 with the ability to determine whether a tube is present at the pipetting position so that thepipetting device 150 may be actuated to aspirate a sample therefrom as appropriate. - The
system 110 supplies the selectable threshold level in digital form to the input of a digital-to-analog converter 560 which latches this value and outputs the same in analog form both to the second input of thecomparator 540 as well as to a voltage controlled gain input terminal of the voltage controlledcurrent amplifier 510. The foregoing arrangement permits thesystem 110 to control the sensitivity of the tubepresence detector system 480 through a relatively wide dynamic operating range. That is, since both the gain of theamplifier 510 as well as the threshold level of thecomparator 540 are controlled by the same signal supplied by theDAC 560, the sensitivity of the system is proportional to the square of the DAC output so that the system's dynamic range is extended as compared with the dynamic range of theDAC 560 output. - By providing the
system 110 with the ability to control the sensitivity of thesystem 480, it is possible for thesystem 480 to be adjusted to reliably detect the presence of thetubes 142 even though tubes of different colors and materials may be employed which may reflect different amounts of light and even though variations in the positions and dispositions of thetubes 142 may be encountered. In addition, by adjusting the sensitivity of thesystem 480 by means of a digital output from thesystem 110, thesystem 480 is easily calibrated to compensate for IRED's 490 andphotodiodes 500 having different characteristics, as well as to compensate for the effects of aging in these components. - Following is a description of an exemplary calibration technique of the
system 480. In accordance with the technique,tubes 142 are placed in a number of specified positions in thecarousel 140 and the carousel is advanced both to positions where tubes are known to be present as well as positions where it is known that no tubes are present. At each such position, thesystem 110 supplies a digital ramp signal to theDAC 560 and stores the value thereof at which the flip-flop 550 toggles, this value being referred to as a "calibration threshold". A detection threshold for use in detecting the presence of atube 142 in normal operation is derived by taking the average of the two calibration thresholds constituting (1) the lowest calibration threshold obtained for the positions at which a tube is present, and (2) the highest calibration threshold obtained for the positions in which a tube is not present. Subsequently, in normal operation thesystem 110 writes the detection threshold into theDAC 560 for use by thesystem 480 for detecting tube presence in normal operation. - With reference now to
Figure 10 , thefluid handling system 90 ofFigure. 1 is illustrated in greater detail therein in combination with theflow cell 50 and aheater block 570 of thesample fluid heater 70 ofFigure 1 . As shown inFigure 10 , aclosed container 580 containing a cleaning fluid is coupled by theline 100 of thefluid handling system 90 to afurther heater system 590, while a further closedcontainer 600 containing assay buffer is coupled by theinlet line 101 to theheater system 590. Theheater system 590 serves to raise the temperatures of the cleaning fluid and assay buffer supplied from thecontainers flow cell 50 as well as the remainder of thefluid handling system 90 preceding theflow cell 50 to assist in achieving temperature control of sample fluids subjected ECL tests in theflow cell 50. The operation of theheater systems - After controlled heating by the
system 590, the cleaning fluid is supplied via aline 610 to a first inlet of amanifold valve 620, while afurther line 630 conducts the assay buffer to a second inlet of themanifold valve 620. Athird line 640 open to the air is connected with a third inlet of themanifold valve 620. Themanifold valve 620 is solenoid actuated and is operative in response to control signals received thereby to select one of the cleaning fluid, assay buffer and air to be supplied to an outlet thereof coupled with amanifold outlet line 650. - With reference also to
Figure 11 , certain elements of thepipetting device 150 are illustrated therein. The pipetting device includes avalve block 660 shown inFigure 10 and illustrated in cross-section inFigure 11 . Aninlet 670 of thevalve block 660 is coupled with themanifold outlet line 650 to receive the fluid supplied by themanifold valve 620. Thepipetting device 150 includes aprobe 680 slidably mounted with respect to thevalve block 660 so that the same may be lowered into arespective holder tube 142 to remove liquid therefrom or, in the alternative, raised to a retracted position as shown inFigure 11 . Theprobe 680 is affixed to acoupling 690 shown in partial cross-section as having a fitting to receive a pipetting device outlet line 700 (Figure 10 ). Thecoupling 690 is mounted on aslidable block 710 slidably mounted on ashaft 720 acting as a linear bearing to guide theblock 710 and attachedprobe 680 as the same is raised and lowered.
Theblock 710 is fitted with a threaded aperture (not shown for purposes of simplicity and clarity) mated with the threads of alead screw 730. Thelead screw 730 is rotatably coupled with a stepper motor (not shown for purposes of simplicity and clarity) which is controllably operable to rotate thelead screw 730 in either of two selectable directions to controllably raise or lower theblock 710 and the attachedpipetting probe 680. Thelead screw 730 andshaft 720 are supported by abase 732. - The
pipetting probe 680 is slidably received in a fitting 740 mated with thevalue block 660 to provide a fluid tight seal therebetween. When theslidable block 710 is fully retracted to its uppermost position as shown inFigure 11 , a poppet mechanism (not shown for purposes of simplicity and clarity) coupled with a spring mountedseal 750 engages theseal 750 in alower opening 760 of thevalve block 660 to form a fluid tight seal therewith. In the disposition as shown inFigure 11 , fluids may be conveyed via the manifold outlet line through thevalve block inlet 670 to theprobe 680 in order to convey cleaning fluid, assay buffer and/or air to the portion of thefluid handling system 90 downstream of thevalve block 660 through theoutlet line 700. In addition, cleaning fluid admitted to the valve block through theinlet 670 serves to clean the lower portion of theprobe 680. - When the
slidable block 710 is lowered by appropriately rotating thelead screw 730, the poppet mechanism is actuated to withdraw theseal 750 from thelower opening 760 to permit theprobe 680 to descend from thevalve block 660 into a respective one of theholder tubes 142 to aspirate fluid therefrom. When this occurs, a pair of O-ring seals valve block 660 by awasher 770 affixed to theprobe 680 to form a fluid tight seal therewith. - The
outlet line 700 is coupled with a T-junction 780 having a first outlet coupled with aline 790 through which fluids are conveyed to theheater block 570 of the sample fluid heater system 70 (Figure 1 ) to be heated thereby in order to bring the fluids conveyed via theline 790 substantially to a predetermined temperature for the conduct of an ECL measurement by theflow cell 50 which receives the heated fluid from anoutlet line 802 of theheater block 570. Fluid received at an inlet of theflow cell 50 from theoutlet line 802 is ultimately conveyed via an outlet thereof to a first inlet of abypass valve 810, a second inlet of thebypass valve 810 being coupled with a second outlet of the T-junction 780. Thebypass valve 810 is a solenoid valve operative to couple either the outlet of theflow cell 50 or the second outlet of the T-junction 780 to anoutlet line 820 of thebypass valve 810. Theoutlet line 820 is coupled with an inlet of aperistaltic pump 830 which serves to controllably draw fluids through thefluid handling system 90,heater block 570 and flowcell 50. An outlet of theperistaltic pump 830 is coupled with awaste fluid container 840 for disposal of used fluids. - The
flow cell 50 is mounted within an environmentally controlledhousing 850, illustrated inFigure 12 . Thehousing 850 has a photomultiplier tube (PMT) 860 of thelight detector system 60 mounted on an upper surface of thehousing 850 and positioned to receive light produced through electrochemiluminescence in theflow cell 50 mounted beneath thePMT 860 within thehousing 850. In order to reduce levels of background light which may interfere with the operation of thePMT 860, thehousing 850 is sealed against stray light on all sides as well as at all openings, for example, where thePMT 860 is mated to thehousing 850. Thehousing 850 is also insulated against heat conduction therethrough by an insulatingcover 870 shown partially broken away for ease of illustration. The temperature within thehousing 850 is controlled by means of the flow celltemperature control system 80 ofFigure 1 which serves to apply heat to the exterior of thehousing 850 to maintain its interior temperature substantially at a predetermined value by means offoil heaters 880 adhesively affixed to three lateral sides of thehousing 850 as well as to a bottom surface thereof. Further details of the flow cell temperature control system will be explained hereinbelow. - The
heater block 570 is mounted on an exterior lateral surface of thehousing 850 and is fabricated of a metal, such as brass, providing good heat conductivity. As shown inFigure 12 , theline 790 through which sample fluids, as well as cleaning fluids, assay buffers and air pass on their way to theflow cell 50 are conducted through theheater block 800 to adjust their temperatures to within at least a predetermined range of temperatures to permit the conduct of ECL tests on the sample fluids in a reproducible manner. As shown inFigure 12 , the fluids emitted from theheater block 570 are conveyed via theline 802 through thehousing 850 and, as shown inFigure 10 , to theflow cell 50. Atemperature sensor 890 is affixed to theheater block 800 to produce a signal representing the temperature thereof. In addition, twopower transistors 900 are affixed to the heater block in order to controllably apply heat thereto for maintaining the temperature of the heater block at a desired level. - With reference now to
Figure 13 , a functional block diagram of the samplefluid heater system 70 is illustrated therein. The samplefluid heater system 70 is implemented as a proportional/integral temperature controller in order to provide close correspondence between a desired temperature of theheater block 570 and the actual temperature thereof. A desired or set temperature of theheater block 570 is written to aDAC 910 by the control and signal/data processing system 110 so that the set temperature value in analog form is output by theDAC 910 to a first input of adifference amplifier 920. A second input of thedifference amplifier 920 is provided with the output of thetemperature sensor 890 and thedifference amplifier 920 serves to produce an error voltage representing the difference between the set or desired temperature of theblock 570 and the actual temperature thereof as sensed by thetemperature sensor 890. The error voltage output by thedifference amplifier 920 is supplied to the input of an analog-to-digital converter 930, as well as to a first input of a summingamplifier 940. An output of the summingamplifier 940 is supplied to the input of adriver 950 which serves to provide a controlled heating current to thepower transistors 900 for controllably heating theblock 800. The loop represented by thetemperature sensor 890,difference amplifier 920, summingamplifier 940 anddriver 950 represents a proportional controller loop, that is, a control loop in which the heating current is proportional to the difference between the set temperature and the measured temperature. - In the operation of a practical proportional control loop having a realistic gain (that is, a gain which is sufficiently limited to avoid instability and consequent oscillation), there typically is a steady state error between the measured value (here the actual temperature of the block 570) and the desired value (that is, the set temperature). Consequently, the
system 70 also employs an integral controller loop which is implemented by the analog-to-digital converter 930, the control and signal/data processing system 110 acting as anintegrator 960 as illustrated inFigure 13 , together with a digital-to-analog converter 970 which serves to convert the output of theintegrator 960 to analog form and supply the same to a second input of the summingamplifier 940. In operation, the integral control loop, after each data acquisition from the analog-to-digital converter 930, adds the converted error voltage to an integral term which is stored by thesystem 110. This value is then scaled by an appropriate gain factor and written to theDAC 970 to be output in analog form to the second input of the summingamplifier 940. The output of theDAC 970 serves to substantially eliminate the steady state error associated with the proportional control loop. In certain instances, however, the integral value is modified to accommodate design limitations of the system. That is, when the error voltage is sufficiently large that the proportional control system alone will drive the power transistors at maximum power, the integral term is set by thesystem 110 to zero. In addition, if the integral term becomes sufficiently large that it likewise will drive the power transistors at full power, the amount of the integral is prevented from increasing so that it does not accumulate past a point where it can have any further effect on the temperature of theheater block 570. - With reference now to
Figure 14 , a block diagram of the flow celltemperature control system 80 is illustrated therein. While the flow celltemperature control system 80 ofFigure 1 may be implemented in the manner illustrated inFigure 13 for the samplefluid heater system 70, the system as illustrated inFigure 14 , is implemented entirely by the control and signal/data processing system 110. That is, the flow celltemperature control system 80 includes atemperature sensor 960 mounted on theflow cell 50 within thehousing 850 and is coupled with an analog-to-digital converter 970 which digitizes the output of thetemperature sensor 960 and provides the same to thesystem 110 acting as a control loop processor 980. The control loop processor 980 carries out both the proportional and integral processing functions as performed by thesystem 70 ofFigure 13 (described hereinabove) and outputs a digital value to the input of a digital-to-analog converter (DAC) 990 representing a drive current to be applied to thefoil heaters 880 adhesively affixed to the exterior of thehousing 850 ofFigure 12 . TheDAC 990 converts the drive value to analog form and supplies the same to the input of adriver 1000 which serves to apply a corresponding heating current to thefoil heaters 880. As an alternative to the dual systems ofFigures 13 and14 , in certain applications thesystem 80 ofFigure 14 can be eliminated and thefoil heaters 880 driven instead by thedriver 950 of the system 70 (Figure 13 ). In addition, in place of heating elements, cooling elements may likewise be used to establish a predetermined test temperature. Such cooling elements include, for example thermoelectric coolers and Peltier coolers. A further alternative is to subject the apparatus to a temperature controlled medium, either liquid or gas (such as air). - In the cross-sectional view of the interior of the
housing 850 as shown inFigure 15 , theflow cell 50 is affixed to thehousing 850 and spaced slightly below the upper surface thereof so that light emitted through electrochemiluminescence within theflow cell 50 propagates towards thePMT 860 to be converted thereby to an electrical signal representing an amount of light received thereby. Theflow cell 50 includes anarm 1010 pivotally mounted thereto, the arm having apermanent magnet 1020 affixed thereto so that themagnet 1020 may be pivoted either to a position in the vicinity of a working electrode of theflow cell 50 for use in collecting magnetic particles with bound ECL labels pursuant to a magnetic particle assay, or away from theflow cell 50, for example, when electrochemiluminescence of the ECL labels is induced in order to avoid interference with the operation of thePMT 860. Thearm 1020 is coupled through acoil spring 1030 with an arm of a solenoid operatedlinear actuator 1040. When the solenoid of thelinear actuator 1040 is deenergized, its arm is drawn outwardly by thecoil spring 1030 so that thearm 1010 pivots away from the vicinity of the working electrode, as shown inFigure 15 . When the solenoid of thelinear actuator 1040 is energized, the arm thereof is drawn within the housing of theactuator 1040, thus exerting a force on thecoil spring 1030 which, in turn, rotates thearm 1010 upwardly to bring themagnet 1020 into a position adjacent the working electrode of theflow cell 50. - As also shown in
Figure 15 , a motor drivenfan 1050 is mounted within thehousing 850 and runs continuously to circulate air within thehousing 850 to maintain a substantially uniform temperature throughout its interior. As also shown inFigure 15 , acircuit board 1060 is mounted on theflow cell 50. Thecircuit board 1060 includes circuitry for coupling the working electrode as well as counter electrodes and a reference electrode included in theflow cell 50 with thesystem 110 for the purpose of applying voltage and current to the counter and working electrodes and to measure such voltages and currents as well as a voltage level on the reference electrode.Circuit board 1060 also includes areference LED 1070 which may be energized selectively to emit a controlled amount of light toward thePMT 860 to enable calibration thereof. - The flow cell is now described with reference to
Figures 16-18 . Theflow cell 50 includes amain housing 1080 fabricated of a durable, transparent and chemically inert material which is easy to machine or injection mold to the configuration illustrated inFigures 16-18 . Suitable materials for thehousing 1080 include acrylic and polymethyl methacrylate. Themain housing 1080 has a first lower surface 1090 (Figure 17 ) through which a fluid inlet defined by a threadedcoupling 1100 andcontiguous conduit 1110 are formed in themain housing 1080. As seen inFigure 17 , theconduit 1110 extends from the threadedcoupling 1100 to anupper surface 1120 of themain housing 1080. - A fluid outlet is also formed in the
main housing 1080 and includes a threaded coupling 1130 extending upwardly from a secondlower surface 1140 of themain housing 1080 to afurther conduit 1150 which extends therefrom to theupper surface 1120. An ECL test chamber or container 1174 is formed between theupper surface 1120 of themain housing 1180 and a lower surface of atransparent block 1160 affixed above theupper surface 1120 and separated therefrom by agasket 1170 which defines lateral walls of the chamber 1174. Thegasket 1170 forms a fluid tight seal between theblock 1160 and themain housing 1080, theblock 1160 andgasket 1170 being held to themain housing 1180 by a plurality of fasteners 1180 (Figure 16 ). The chamber 1174 thus defined by themain housing 1080, theblock 1160 and thegasket 1170 communicates with theconduit 1110 adjacent a first lateral side of the chamber 1174 and with theconduit 1150 at a second lateral side of the chamber 1174 opposite the first lateral side. Accordingly, fluids introduced through the fluid inlet defined by thecoupling 1100 andconduit 1110 flow through the chamber 1174 from right to left as viewed inFigure 17 and are emitted therefrom through the fluid outlet formed by theconduit 1150 and threaded coupling 1130, so that the fluid inlet, the chamber and the fluid outlet define a fluid flow path through theflow cell 50. - With reference in particular to
Figures 17 and18 , a workingelectrode 1182 is arranged in a shallow groove formed in theupper surface 1120 of themain housing 1180 and has a longitudinal axis arranged generally transverse to a longitudinal axis of the chamber 1174 extending from the first lateral side thereof to its second lateral side and is positioned laterally centrally thereof between theconduits electrode 1182 is held within the shallow groove in thetop surface 1120 of themain housing 1080 by means of afirst retainer block 1230 held against thesurface 1120 by a pair of fasteners and serving to maintain a first electrical lead (not shown for purposes of simplicity and clarity) in conductive contact with the working electrode for coupling the same with thecircuit board 1060. - With reference particularly to
Figures 16 and17 , afirst counter electrode 1190 is arranged to extend along a bottom surface of theblock 1160 forming an upper surface of the chamber 1174 from a position approximately opposite a first lateral side of the workingelectrode 1182 toward the first lateral side of the chamber 1174 adjacent theconduit 1110 and therebeyond between thegasket 1170 and theblock 1160 and is forward upwardly at a right angle to extend along a first lateral side of theblock 1160. Thefirst counter electrode 1190 is held against the first lateral side of theblock 1160 by asecond retainer block 1210 fastened by a pair of fasteners to theblock 1160 and which also serves to securely connect an electrical lead (not shown for purposes of simplicity and clarity) to thefirst counter electrode 1190 to couple the same with the circuit board 1060 (Figure 15 ). - A second counter electrode 1200 extends along the bottom surface of the
block 1160 from a position approximately opposite a second lateral side of the workingelectrode 1182 outwardly along the top wall of the chamber 1174 formed by the bottom wall of theblock 1160 toward theconduit 1150 and therebeyond between theblock 1160 and thegasket 1170 to a second lateral edge of theblock 1160 where the second counter electrode is formed at a right angle to extend upwardly therealong. Athird retainer block 1220 retains the second counter electrode against the second lateral side of theblock 1160 by means of a further pair of fasteners and serves to securely couple a further electrical lead (not shown for purposes of simplicity and clarity) to the second counter electrode 1200 for coupling the same to thecircuit board 1060. Materials suitable for the workingelectrode 1182 and thecounter electrodes 1190 and 1200 include platinum and gold. - It will be seen especially from
Figure 17 that thecounter electrodes 1190 and 1200 are arranged on a wall (that is, the bottom surface of the block 1160) of the chamber opposite a second wall thereof (that is, theupper surface 1120 of the main housing 1080) on which the workingelectrode 1182 is arranged. Conventional flow cells place the working and counter electrodes on the same wall of the chamber in which ECL is induced so that the emitted light can pass through an opposite transparent wall of the chamber to be detected by a PMT. However, certain electrode materials are prone to flake off as fluid flows by, thus tending to form a conductive bridge which shorts out the counter and working electrodes, thereby rendering the flow cell unusable until the conductive bridge has been removed by cleaning. - The
flow cell 50 as shown particularly inFigure 17 substantially alleviates this problem by positioning a counter electrode on a wall of the ECL chamber opposite a wall thereof on which the working electrode is arranged but positioned so that the surface of the working electrode does not oppose the counter electrode, but rather is arranged opposite a wall which is made of transparent material. Consequently, any material which may flake off either a counter electrode or the working electrode will not tend to form a conductive bridge between the counter and working electrodes, while at the same time light emitted by ECL labels adjacent the surface of the working electrode can be transmitted through the transparent wall to be detected. - A further advantage provided by the
flow cell 50 ofFigures 16-18 is provided by the arrangement ofcounter electrodes 1190 and 1200 on opposite sides of the workingelectrode 1182 which serves to minimize variations in the flow of electric current between thecounter electrodes 1190 and 1200, on the one hand, and the workingelectrode 1182, on the other, which may be caused, for example, by variations in fluid flow or composition within the ECL chamber 1174. - As noted above in connection with
Figure 15 , amagnet 1020 is mounted on anarm 1010 which is pivotally connected with theflow cell 50. With reference in particular toFigures 16 and18 , thearm 1010 andmagnet 1020 are illustrated therein in an upper position in which themagnet 1020 is brought in close proximity to the working electrode, being separated therefrom only by a relativelynarrow wall 1240 of themain housing 1080. In this upper position, themagnet 1020 serves to accumulate magnetic particles bound to ECL labels adjacent a surface of the working electrode exposed to fluids in the ECL chamber 1174 in carrying out magnetic particle assays. Since it is desirable to move themagnet 1020 downwardly away from thePMT 860 when ECL measurements are carried out, thearm 1010 is mounted to themain housing 1080 by apivot pin 1250. - With reference in particular to
Figures 16 and17 , areference electrode 1260 includes, for example, a wire immersed in an ionic solution permanently retained by an outer glass housing capped at an outer end by a glass frit which permits ionic communication between the ionic fluid within the glass housing and fluids which may come in contact with the glass frit. Conventional flow cell structures bring the glass frit of the reference electrode directly in contact with fluids within the fluid flow path, so that ionic exchange takes place therewith and the chemical composition of the ionic fluid within the glass housing of the reference electrode gradually changes so that the electrical characteristics of the reference electrode change or drift disadvantageously over time. - The
flow cell 50 ofFigures 16-18 substantially alleviates this problem by interposing a further ionic fluid between the flow path and the ionic fluid within the reference electrode. Moreover, the second ionic fluid is retained within achamber 1274 formed by themain housing 1080 and alateral block 1270 held to the main housing by a plurality of fasteners and sealed thereagainst by afurther gasket 1280. Theblock 1270 may be made, for example, of the same material as themain housing 1080. As shown inFigure 16 , thereference electrode 1260 is inserted into the chamber formed between theblock 1270 and themain housing 1080 to bring its glass frit into contact with an ionic conductive medium therein. A glass orceramic frit 1290 is positioned in an aperture within themain housing 1080 joining theconduit 1150 and thechamber 1274 and is retained therein by aplug 1300 which presses against an 0-ring seal 1310 to seal the outer periphery of the frit 1290 against invasion of fluids from theconduit 1150 or loss of ionic conductive media within thechamber 1274 to theconduit 1150. - A refill aperture is formed in the
upper surface 1120 of themain housing 1080 extending to thechamber 1274 and is sealed by aremovable plug 1320 which permits an ionic media within thechamber 1274 to be replaced. A suitable ionic conductive medium for filling thechamber 1274 is a gel including sodium chloride and agarose having a concentration selected to render the gel solid at room temperature, but liquefiable at 80°C so that the same may be poured into thechamber 1274 through the aperture in theupper surface 1120 of themain housing 1080. The gel also contains phenolphthalein providing an indicator to detect leaks across thefrit 1290. In particular, the phenolphthalein turns the gel pink when cleaning fluid from theconduit 1150 comes in contact with the gel due to a change in pH of the gel brought about by the cleaning fluid. - As noted hereinabove, the
temperature sensor 960 of the temperature control system ofFigure 14 is mounted on theflow cell 50 within thehousing 850 ofFigure 15 . As shown inFigure 16 , thetemperature sensor 960 is mounted on a side wall of themain housing 1080. -
Figures 19A through 19C provide a block diagram of the control and signal/data processing system 110 of theFigure 1 embodiment. With reference first toFigure 19A , acentral processing unit 1330 including a microprocessor, microcomputer or the like, is bidirectionally coupled with aRS 232serial interface 1340 coupled with serial input/output port 120 for data communication. TheCPU 1330 is also coupled bidirectionally with amemory 1350 including a RAM as well as nonvolatile storage, for example, provided by flash memory circuits. TheCPU 1330 is operative to communicate with an external source of assay control programs through theinterface 1340 to receive and store such programs in thememory 1350. The external programming source may be, for example, a personal computer in which a user inputs programs through a keyboard, disk drive, or other input device. As explained hereinbelow, the control and signal/data processing unit 110 is operative to receive and store a plurality of assay control programs, as well as to run such programs simultaneously to provide a multitasking capability which promotes efficient use of the apparatus. In addition, by permitting a user to create and run multiple programs, it is possible for the user to design and test relatively small portions of a more complex assay which greatly facilitates assay development. - The
CPU 1330 is also bidirectionally coupled with a timer processor unit (TPU) 1360 in the form of a programmable timer device operative to generate and read clock signals. As explained in greater detail hereinbelow, the TPU 1360 is employed to generate stepper motor drive signals as well as to convert voltage-to-frequency converted values to a digital form which may be processed by theCPU 1330. TheCPU 1330 is also bidirectionally coupled with an input/output unit 1370 which provides a digital communication capability between theCPU 1330 and various peripheral detection and drive circuits, as explained in greater detail below. - With reference also to
Figure 19B , the input/output unit 1370 is bidirectionally coupled with a digital input/output circuit 1380 which serves both as a digital multiplexer and dimultiplexer for digital signals provided from the input/output unit 1370 and various peripheral digital circuits, as well as for buffering various digital signals to be communicated to and from the peripheral circuits. As shown inFigure 19B , the digital input/output unit 1380 is coupled with a steppermotor control circuit 1390, which is also coupled with the TPU 1360 to receive a stepper motor step pulse signal. The steppermotor control circuit 1390 serves to buffer direction and enable signals generated by theCPU 1330 and supplied via the digital input/output circuit 1380 for use in generating appropriate control signals to drive a selected one of the stepper motors of the apparatus selectably in high or low power mode and in normal or reverse direction. Thestepper motor circuit 1390 includes separate latches to store direction and enable signals for each of the carousel rotating motor 390 (Figure 6 ), the peristaltic pump 830 (Figure 10 ), the linear actuator 1040 (Figure 15 ) and the probe up/down drive motor described in connection withFigure 11 . The stepper motor control circuit is coupled with an optical interface and drivecircuit 1400 to provide the various control signals for controlling the various stepper motors. The optical interface and drivecircuit 1400 serves to decouple voltage spikes generated by the stepper motors from the remainder of thesystem 110 as well as to generate the necessary drive signals. - The
circuit 1380 is also coupled with a valve driver circuit 1410 which serves to latch control signals for controlling the states of the solenoids in the manifold 620 as well as the state of the solenoid controlling thebypass valve 810, both as illustrated and described in connection withFigure 10 . The circuit 1410 likewise includes suitable driver circuits for driving the valve solenoids in accordance with the latched control signals. In particular, the manifold 620 includes three valves, each controlled by a respective solenoid for either communicating or blocking access from a respective inlet of the manifold 620 to theoutlet line 650 thereof. - The
circuit 1380 also receives the output of the D-type flip-flop 550 of the tube presence detection system ofFigure 9 and latches the same to be provided to theCPU 1330 for detecting the presence of aholder tube 142 at the pipetting position. Thecircuit 1380 has a plurality ofinputs 1430 for receiving temperature detection signals from the various temperature control systems described hereinabove. In addition, thecircuit 1380 has an input coupled with an opticalswitch interface circuit 1440 which, in turn, receives detection signals from theoptical interrupter 472 ofFigure 9 (for detecting the homing position of the carousel 140), a home sensor for theprobe 680 ofFigure 11 , a home sensor for thepump 830 ofFigure 10 and a sensor providing a signal indicating whether theexterior housing 130 has been opened, in order to provide the system with the ability to shut down high voltage supplies in that event. Theinterface circuit 1440 conditions the detection signals from the various optical interruptors and latches the same for providing appropriate outputs to thecircuit 1380 for provision to theCPU 1330 for control purposes. - The digital input/
output circuit 1380 has a serial output coupled with a serial input of a control digital-to-analog converter 1450 which serves to latch digital values provided by thecircuit 1380 and convert the same to analog form for carrying out various control functions described in greater detail hereinbelow. More particularly, thecontrol DAC 1450 latches a threshold level signal for tube presence detection and converts the same to analog form which it supplies over anoutput 1460 to thecomparator 540 ofFigure 9 (so that thecontrol DAC 1450 implements the function of theDAC 560 as shown inFigure 9 ). In addition, thecontrol DAC 1450 latches a digital value representing a drive voltage for theagitation motor 250 and outputs the same in analog form to an agitationmotor drive circuit 1470 which, in turn, provides a driving current to themotor 250. Thecontrol DAC 1450 also latches set temperatures for each of thetemperature control systems Figure 1 as well as thesystem 590 ofFigure 10 and outputs the same in analog form over a plurality of output lines indicated as 1480 inFigure 19B . Finally, thecontrol DAC 1450 latches a digital value received fromcircuit 1380 representing a high voltage level to be applied to thePMT 860 and converts the same to analog form which it supplies to a PMT highvoltage power supply 1490 for controlling the high voltage applied thereby to thePMT 860. - Referring also to
Figure 19C , the digital input/output circuit 1380 outputs digital values representing reference LED drive level and waveform generation parameters to a digital-to-analog converter (DAC) 1500 having a plurality of addressable latches for storing these values to be supplied respectively to a referenceLED drive circuit 1510 for supplying an appropriate drive level to thereference LED 1070 ofFigure 15 and to awaveform generator 1520 which serves to generate waveforms appropriate for driving the electrodes of theflow cell 50 for carrying out ECL measurements, as well as for cleaning and conditioning the electrodes. TheDAC 1500 also receives a reference voltage level from avoltage reference circuit 1530. - In response to the analog values received from the
DAC 1500, thewaveform generator 1520 selectably generates either a ramp voltage waveform having a slope endpoint specified by the value supplied by theDAC 1500 or else a specified, constant output voltage. The waveforms thus produced by thewaveform generator 1520 are supplied to an input of apotentiostat 1540. Thepotentiostat 1540 is coupled with each of the reference, counter and working electrodes and serves to apply the waveform received from thewaveform generator 1520 so that the voltage level appearing at the reference electrode corresponds with the voltage output by thewaveform generator 1520. Since the reference electrode does not conduct current, it will be seen with reference toFigure 17 that the reference electrode will have a voltage level which is essentially the same as the voltage level on the counter electrode 1200. In addition, the counter electrode 1200 is coupled with thecounter electrode 1190 on thecircuit board 1060, so that the voltage level at thecounter electrode 1190 is the same as that at the counter electrode 1200. Moreover, until current begins to flow between the counter electrodes and the workingelectrode 1182, the voltage level at the workingelectrode 1182 will be essentially the same as that on the counter electrodes and the reference electrode. However, once current begins to flow from the counter electrodes to the working electrode in response to a drive voltage applied between the counter and working electrodes by thepotentiostat 1540, the voltage level at the surface of the working electrode falls below that of the counter and reference electrodes in proportion to the amount of current flowing between the counter and working electrodes. This provides the advantage of reducing the slope in the voltage waveform at the surface of the working electrode which leads to an improvement in measurement sensitivity. Further details of the operation of thewaveform generator 1520 and thepotentiostat 1540 may be obtained with reference toU.S. Patent No. 5,068,088 issued November 26, 1991 entitled Method and Apparatus for Conducting Electrochemiluminescent Measurements. - The
potentiostat 1540 produces a current sensing voltage representing current flowing between the counter and working electrodes, as well as values representing electrode.voltage levels and supplies these signal in analog form to a first input of a multiplexer and voltage-to-frequency converter 1550 having a plurality of inputs at which it receives respective analog voltages to be multiplexed and converted to signals in the frequency domain which, in turn, it supplies to the digital input/output circuit 1380 and timer processor unit 1360 for conversion to a form suitable for processing by theCPU 1330. Thecircuit 1550 also receives the output of thewaveform generator 1520, the reference voltage from thecircuit 1530 and a temperature detection signal from the temperature sensor 960 (Figure 16 ) for multiplexing and conversion in the same manner as the signals received from thepotentiostat 1540. Aluminometer 1560 receives the output of thePMT 860 and provides both a low gain output on anoutput terminal 1562 and a high gain output on anoutput line 1564 each of which is coupled with a respective input of the multiplexer and voltage-to-frequency converter 1550. The provision of low and high gain outputs from theluminometer 1560 provides a wide dynamic range of operation for the apparatus. Finally, thecircuit 1550 has an input coupled to receive a ground level reference input. -
Figure 20 provides a diagram illustrating the functional relationships among basic program elements of the software which controls the operation of theCPU 1330 ofFigure 19A . Thesystem 110 employs amultitasking operating system 1570 on which asupervisor program 1580 runs for managing the overall operation of thesystem 110 and, therefore, the apparatus overall. The software also includesbinary sequences 1590 each of which may be called by a higher level command to carry out a relatively specific, predefined task. Also included are a plurality ofsequence engines 1600 each of which operates independently of the other sequence engines and acts as an interpreter for higher level commands, calling thebinary sequences 1590 as appropriate to execute these commands. A number ofdevice drivers 1610 execute commands which control instrument hardware, such as valves, stepper motors, and the like by outputting appropriate digital control signals via the input/output unit 1370 ofFigure 19A . Thedevice drivers 1610 also control the storage of newly received assay control programs in non-volatile memory included within thememory block 1350 ofFigure 19A . Finally, adata link controller 1620 manages the data communications via theRS232 interface 1340, including activities such as packeting, routing and error checking. - The supervisor program serves to initialize the system, including setting up the
device drivers 1610, initializing hardware and starting the data link controller task. In addition, the supervisor program starts asequence engine 1600 in response to a command received by the serial input/output port 120 and then assumes a background status, awaiting an event requiring its intervention. Such events include, for example, a system error, a request for a system reset, and a failure of the data link, in which case some or all of the system may require initialization by the supervisor program. In addition, if the supervisor has initiated a plurality ofsequence engines 1600 to run simultaneously, it serves to keep track of the system conditions overall and responds to any conflict between instructions carried out by different sequence engines in order to resolve the same. - The following Table I provides a summary of commands available to a user for programming the operation of the embodiment of
Figure 1 , which may be entered via the serial input/output port 120 individually or in the form of an assay control program which is stored and selectably run by the control and signal/data processing system 110.TABLE I SUMMARY OF USER PROGRAMMABLE COMMANDS COMMAND DESCRIPTION Acquire Start or stop capturing data representing either ECL luminosity (temperature compensated), dark current level, or reference level (LED reference 1070 on) Carousel Homes the carousel 140 or moves it to next tube position, as selected Carpos Moves the carousel 140 to a specified tube position Cello Turns off electrical supply to flow cell 50 by opening feedback loop of potentiostat 1540 and shorting its output Heater Sets a specified temperature for a selected one of the heating systems 70, 80 or 590 Idel Initiates a specified apparatus (instrument) delay Ireset Resets apparatus Lumref Turns on reference LED 1070 for calibration of PMT 860 Magnet Moves magnet 1020 up or down PMT Sets PMT 860 high voltage (to select PMT sensitivity according to requirements of selected assay) Probe Moves probe 680 up or down, as selected Ptog Changes the polarity of the waveform produced by the waveform generator 1520 and enables potentiostat 1540 Pump Homes peristaltic pump 830, turns the pump on at a specified speed and direction, or turns off the pump, as selected Ramp Commands waveform generator 1520 to generate a ramp voltage waveform having a specified end point and slope, and enables potentiostat 1540 Program Store Stores assay control program received via RS232 interface Valve Turns a specified valve on or off, as selected Volt Commands waveform generator to output a constant, specified voltage level, and enables potentiostat 1540 Vortex Sets rotational speed of agitation motor 250 (from zero to a maximum value) - Most of the commands summarized in Table I are followed by an argument providing further information necessary for carrying out the command, such as a device to be operated, a device state (speed, direction, on/off state, position) or signal to be generated. For example, the Acquire command requires an argument specifying the type of data to be captured while the Heater command requires an argument specifying the
particular heating system memory 1350 stores a table of values which specify the amount of an adjustment which must be made in a given ECL luminosity reading depending on the deviation of the actual sample fluid temperature from a nominal testing temperature. In other words, the memory means stores data representing temperature dependence of light produced through electrochemiluminescence. In carrying out the temperature compensation routine, the temperature measured by thetemperature sensor 960 mounted onflow cell 50 is employed to access the appropriate compensation data for this purpose. - Following is a description of an exemplary magnetic particle ECL assay for obtaining a number of ECL measurements for samples presented in
respective holder tubes 142 mounted by a user in the carousel 140 (Figure 4 ). It will be appreciated that, since the apparatus is programmable, further assays may be carried out thereby which differ substantially from the described exemplary assay, but which still employ the features of the present invention and, thus, are within the scope of the claims hereof. -
Figures 21A and21B provide a flow chart of a main processing loop of the exemplary assay. With reference first toFigure 21A , after processing has begun, the apparatus is initialized and assay parameters are specified, as indicated instep 1630. That is, the apparatus is reset by the Ireset command, followed by the specification of the parameters, the number ofholder tubes 142 to be sampled in the course of the assay and the carrying out of a system cleaning subroutine Clean-line 1640.Figures 22A and22B provide a flow chart of the Clean-line subroutine. When called, the Clean-line subroutine proceeds to actuate the cleaning fluid solenoid valve of the manifold to switch cleaning fluid to theoutlet line 650 of the manifold 620, as indicated instep 1650 with the use of the Valve command summarized in Table I. In the followingstep 1660 the peristaltic pump is started by means of the Pump command and in a subsequent step 1670 a constant voltage level Vc of the waveform generator is produced by means of the Volt command to set the voltage of theflow cell 50 substantially at the level Vc in order to draw a cleaning fluid through theflow cell 50 at a predetermined cleaning voltage. - Thereafter in a
step 1680, the bypass valve is turned on and off a predetermined number (N) of times by means of the Valve command in order to expel foreign material which may have become trapped in the T junction 780 (Figure 10 ). Subsequently to thestep 1680, in astep 1690, the manifold air inlet is turned on and off N times which serves to inject slugs of airs into the system (while the pump remains on) to mechanically dislodge particulate matter which is then carried away by the cleaning fluid. In the followingstep 1700, the assay buffer valve of the manifold is turned on (while the cleaning solution valve is turned off) to introduce assay buffer into the system. The clean line subroutine is concluded by stopping the pump in thestep 1730. - Upon return to the main loop as illustrated in
Figures 21A and21B , the program begins executing a programloop including steps 1740 through 1790 repeatedly until all of the sample tubes have been read. In the repeated loop, the carousel is first moved to the position of the next tube by means of the Carousel command instep 1740. Thereafter aStartube subroutine 1750 is called. - With reference now to
Figures 23A and 23B , the Startube subroutine serves to draw a sample fluid from aholder tube 142 at the pipetting position. Pursuant to the Startube subroutine, in astep 1800 the pump is brought to a home position in order to permit a precise amount of the sample to be withdrawn from the tube. In asubsequent step 1810 the vortexing motor is turned on by the Vortex command and thereafter the assay buffer valve of the manifold is opened by the Valve command in astep 1820. In thesubsequent step 1830, the pump is turned on to draw the assay buffer through the flow cell while a succession of voltage ramps is applied in thestep 1840 to condition the working electrode by bringing it into a reproducible electrochemical condition by either removing or forming an oxide layer at its working surface. Thereafter the voltage is maintained at a preset value in order to apply a predetermined constant potential to the working electrode, so that the working electrode is conditioned to ensure reproducible test results. - Thereafter, the vortexing motor is turned off (step 1850), the pump is turned off and returned to its home position (step 1860) and the assay buffer valve is closed (step 1870), in preparation to aspirate the sample from the holder tube. Once these steps have been accomplished, the
probe 680 is lowered into the holder tube pursuant to the Probe command instep 1880, and the magnet is brought to its up position in astep 1890 pursuant to the Magnet command in order to attract magnetic particles with bound ECL labels to the surface of the working electrode when the sample fluid enters theflow cell 50. Then the sample is drawn into the probe in a succession ofsteps step 1910 at the end of which the pump is turned off in astep 1920. When the pump is turned off, a precisely measured amount of the sample has been drawn into theprobe 680 which is then withdrawn from the sample tube by means of the Probe command, as indicated in astep 1930 and processing returns to the main processing loop ofFigures 21A and21B . - Upon return to the main loop, the program calls a Trans subroutine in a
step 1760 during which the sample fluid is drawn through theflow cell 50 at a controlled rate for the purpose of accumulating the magnetic particles in the fluid adjacent the working electrode in a controlled manner to ensure reproducibility of the test results. Pursuant to the Trans subroutine, as illustrated inFigure 24 , the assay buffer valve of the manifold is turned on to supply assay buffer to theoutlet line 650 thereof in astep 1940. In asubsequent step 1950 the pump is turned on to draw assay buffer into the system for a predetermined period of time and at a controlled rate so that the sample fluid which precedes the assay buffer in the fluid transfer system is controllably drawn through theflow cell 50, as mentioned above, followed by assay buffer to remove sample particles which have not been captured by the magnet at the surface of the working electrode. Thereafter in astep 1960 the cleaning fluid valve of the manifold is turned on (and the assay buffer valve turned off) for a predetermined period of time to introduce cleaning fluid into the system, although not yet into theflow cell 50. At the end of the predetermined period of time, the pump is turned off in astep 1970, all of the manifold valves are closed (step 1980) and the magnet is moved to the down position (step 1990) in preparation for carrying out the ECL measurement, whereupon the program returns once again to the main loop. Upon return to the main loop, a Measure subroutine (step 1770) is called for carrying out the ECL measurement. With reference toFigure 25 in which the Measure subroutine is summarized, a dark current level of thePMT 860 is first obtained in a step 2000 (which actually represents three commands, namely, Acquire dark current level on, followed by Idel for a predetermined time period, and then Acquire dark current level off). Subsequently, a further Acquire command is executed to commence ECL data capture in astep 2010, whereupon a suitable sequence of ramp voltage waveforms are applied to theflow cell 50, as indicated in astep 2020, in order to controllably induce electrochemiluminescence by the sample fluid in theflow cell 50. After a predetermined period of time, the Acquire command is again executed to end ECL data capture, as indicated instep 2030. Once the ECL data capture task has been completed, the flow cell voltage is set at zero (step 2040), an excitation voltage is applied to the reference LED by executing the Lumref command (step 2050) and an Acquire sequence (step 2060) is carried out to capture PMT readings to provide a reference for evaluating the operating state of the PMT. Once the reference data has been captured, the reference LED is turned off (step 2070) and the program returns once again to the main loop. Following the Measure subroutine, the Clean-line subroutine (step 1780) is again carried out and, in thesubsequent step 1790, it is determined whether all of the sample tubes have been read. If not, the program returns to thestep 1740 to begin a further measurement sequence to measure the sample contents of the next holder tube. - Once all of the measurements have been carried out pursuant to the exemplary assay, the program in
step 1790 proceeds to astep 2080 in which the various apparatus devices are turned off, followed by astep 2090 in which the various valves of the apparatus are closed, after which the control and signal/data processing system 110 is brought to a stand-by condition instep 2100 to complete the assay. - It will be appreciated that various elements of the present invention may be implemented in whole or in part using either analog or digital circuitry and that all or part of the control functions as well as the signal and data processing functions thereof may be carried out either by hardwired circuits or with the use of a microprocessor, microcomputer or the like.
- Although specific embodiments of the invention have been described in detail herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope of the invention as defined in the appended claims.
Claims (12)
- An apparatus for use in carrying out electrochemiluminescence test measurements, comprising:a flow cell (50) for containing an electrochemiluminescent fluid sample; the flow cell having a fluid inlet (1100, 1110) to receive the electrochemiluminescent fluid sample and a fluid outlet (1130, 1150) to conduct the electrochemiluminescent fluid sample from the flow cell (50); and further comprising a fluid transport system (802, 820) coupled with the fluid inlet (1100, 1110) and the fluid outlet (1130, 1150) for conducting the fluid sample to the fluid inlet (1100, 1110) and conducting the fluid sample from the fluid outlet (1130, 1150);a working electrode (1182) having an electrode surface within the flow cell;a supply of electrical energy coupled with the working electrode (1182) for supplying electrical energy to the electrochemiluminescent fluid sample within the flow cell (50);an output signal producing means (860) for producing an output signal representing a detected value based on light produced through electrochemiluminescence of the fluid sample within the flow cell (50);and wherein the apparatus is characterised by a temperature effect adjustment means (70; 80), which, in use, produces an electrochemiluminescence temperature effect adjusted output signal, wherein said temperature effect adjustment means is for at least one of:a) adjusting the temperature of the electrochemiluminescent fluid sample to a value within a predetermined range of temperature values as the electrochemiluminescent fluid sample is conducted to the flow cell by the fluid transport system; andb) adjusting the output signal based on the temperature of the fluid sample; andc) establishing a temperature of the flow cell within the predetermined range of temperature values.
- The apparatus of Claim 1, wherein the temperature effect adjustment means (70; 80) is operative to adjust the temperature of the fluid sample to a value within a predetermined range of temperature values.
- The apparatus of Claim 2, wherein the temperature effect adjustment means (70; 80) is operative to substantially establish a predetermined temperature of the fluid sample within the predetermined range of temperature values.
- The apparatus of Claim 2, wherein the temperature effect adjustment means (70; 80) is operative to heat the fluid sample to a value within the predetermined range of temperature values.
- The apparatus of Claim 2, wherein the temperature effect adjustment means (70; 80) is operative to cool the fluid sample to a value within the predetermined range of temperature values.
- The apparatus of Claim 1, wherein the temperature effect adjustment means (70; 80) is operative to adjust the output signal based on the temperature of the electrochemiluminescent fluid sample to produce a temperature effect adjusted output signal.
- The apparatus of Claim 6, wherein the temperature effect adjustment means (70; 80) comprises memory means (110) storing data representing temperature dependence of light produced through electrochemiluminescence, and means (890, 920, 940, 950; 930, 960, 970, 940; 960, 970, 980, 990, 1000; 1050) for adjusting the output signal based on the stored data.
- The apparatus of Claim 1, wherein:-said flow cell (50) comprises a flow cell housing (1080) defining a sample chamber (1174), formed between an upper surface (1120) of the main housing (1080) and a lower surface of a transparent block (1160) affixed above the upper surface (1120), for holding said electrochemiluminescent fluid sample, the flow cell housing (1080) having the fluid inlet defined by a coupling (1100) and a contiguous conduit (1110) formed through a first lower surface (1090) of the main housing (1080), for introducing the electrochemiluminescent fluid sample to the sample chamber (1174) and the fluid outlet defined by a coupling (1130) extending upwardly from a second lower surface (1140) of the main housing (1080) to a further conduit (1150) which extends therefrom to the upper surface (1120), for emitting the fluid sample from the sample chamber (1174) and the fluid outlet (1130, 1150) defining a fluid flow path through the flow cell housing (1080); said working electrode (1182) and a counter electrode (1190; 1200) are disposed within the same container (1174) for exposure to the electrochemiluminescent fluid sample therein;said flow cell housing (1080) defining an ionic medium container (1274) coupled with the fluid flow path to permit ionic exchange therebetween; and a reference electrode (1260) that is disposed within the ionic medium container (1274) for exposure to ionic media therein; andwherein the ionic medium container (1274) is disposed within the flow cell housing (1080) in a chamber formed by the main housing (1080) and a lateral block (1270) held to the main housing.
- The apparatus of Claim 1, wherein:said flow cell (50) comprises a sample chamber (1174) having a fluid flow path therethrough, the sample chamber (1174) having the fluid inlet defined by a coupling (1100) and a contiguous conduit (1110) formed through a first lower surface (1090) of the main housing (1080), for introducing an electrochemiluminescent fluid sample to the fluid flow path and the fluid outlet defined by a coupling (1130) extending upwardly form a second lower surface (1140) of the main housing (1080) to a further conduit (1150) which extends therefrom to the upper surface (1120) for emitting the fluid sample from the fluid flow path, for conducting fluids through the sample chamber (1174) along a flow direction through the fluid flow path from the fluid inlet (1100, 1110) to the fluid outlet (1130, 1150);said electrode surface of the working electrode (1182) is disposed to one side of the fluid flow path and the sample chamber (1174) enables transmission of light therethrough produced by electrochemiluminescence of the fluid sample adjacent the working electrode surface; andsaid apparatus further comprising a counter electrode means (1190; 1200) for providing an electric current through the fluid sample to the working electrode surface, the counter electrode means (1190; 1200) including a first portion (1190) disposed upstream of the working electrode surface and a second portion (1200) disposed downstream of the working electrode surface.
- The apparatus of Claim 1, wherein:said flow cell (50) comprises a sample chamber (1174) formed between an upper surface (1120) of the main housing (1080) and a lower surface of a transparent block (1160) affixed above the upper surface (1120), having a fluid flow path therethrough, the sample chamber (1174) having the fluid inlet (1100, 1110) defined by a coupling (1100) and a contiguous conduit (1110) for introducing a fluid sample to the fluid flow path and the fluid outlet defined by a coupling (1130) extending upwardly from a second lower surface (1140) of the main housing (1080) to a further conduit (1150) which extends therefrom to the upper surface (1120) for emitting the fluid sample from the fluid flow path, for conducting fluids through the sample chamber (1174) along a flow direction through the fluid flow path from the fluid inlet (1100, 1110) to the fluid outlet (1130, 1150);said apparatus further comprises a first counter electrode (1190; 1200) having an electrode surface exposed to fluids within the fluid flow path;said working electrode (1182) is positioned within the sample chamber (1174) and having an electrode surface exposed to fluids within the fluid flow path; andsaid electrode surface of the working electrode (1182) is displaced from the electrode surface of the first counter electrode (1190; 1200) laterally with respect to the flow direction of fluid within the fluid flow path such that conductive material entering within the fluid flow path from the electrode surface of one of the first counter electrode (1190; 1200) and the working electrode (1182) does not form a conductive bridge with the other of the first counter electrode (1190; 1200) and the working electrode (1182).
- The apparatus of Claim 10, wherein at least a portion of the electrode surface of the first counter electrode (1190) is positioned upstream of the electrode surface of the working electrode (1182); and
wherein the apparatus further comprises a second counter electrode (1200) positioned within the sample chamber (1174) and having an electrode surface exposed to fluids within the fluid flow path;
the electrode surface of the second counter electrode (1200) being displaced laterally with respect to the electrode surface of the working electrode (1190), at least a portion of the electrode surface of the second counter electrode (1200) being positioned downstream of the electrode surface of the working electrode (1182). - A method for carrying out electrochemiluminescence test measurement, comprising introducing a fluid sample into a cell (50) of an apparatus according to any of Claims 1 to 11, supplying electrical energy to the fluid sample through the working electrode (1182) to induce electrochemiluminescence, and detecting said electrochemiluminescence.
Applications Claiming Priority (1)
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PCT/US1994/009124 WO1996005501A1 (en) | 1993-05-14 | 1994-08-15 | Apparatus and methods for carrying out electrochemiluminescence test measurements |
Publications (3)
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EP0783681A1 EP0783681A1 (en) | 1997-07-16 |
EP0783681A4 EP0783681A4 (en) | 1998-11-25 |
EP0783681B1 true EP0783681B1 (en) | 2008-10-29 |
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EP94925843A Expired - Lifetime EP0783681B1 (en) | 1994-08-15 | 1994-08-15 | Apparatus and method for carrying out electrochemiluminescence test measurements |
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EP (1) | EP0783681B1 (en) |
JP (1) | JPH10504105A (en) |
KR (1) | KR100358448B1 (en) |
AT (1) | ATE412887T1 (en) |
AU (1) | AU706107B2 (en) |
CA (1) | CA2197662C (en) |
DE (1) | DE69435159D1 (en) |
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HK (1) | HK1001415A1 (en) |
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US4328185A (en) * | 1980-06-26 | 1982-05-04 | Boehringer Mannheim Corporation | Automated chemical testing apparatus |
US5147806A (en) * | 1988-04-29 | 1992-09-15 | Igen, Inc. | Method and apparatus for conducting electrochemiluminescence measurements |
US5068088A (en) * | 1988-11-03 | 1991-11-26 | Igen, Inc. | Method and apparatus for conducting electrochemiluminescent measurements |
US5061445A (en) * | 1988-11-03 | 1991-10-29 | Igen, Inc. | Apparatus for conducting measurements of electrochemiluminescent phenomena |
GB9119382D0 (en) * | 1991-09-11 | 1991-10-23 | Knight Scient Ltd | Apparatus for monitoring liquids |
US5466416A (en) * | 1993-05-14 | 1995-11-14 | Ghaed; Ali | Apparatus and methods for carrying out electrochemiluminescence test measurements |
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1994
- 1994-08-15 EP EP94925843A patent/EP0783681B1/en not_active Expired - Lifetime
- 1994-08-15 JP JP8507255A patent/JPH10504105A/en active Pending
- 1994-08-15 AU AU75626/94A patent/AU706107B2/en not_active Ceased
- 1994-08-15 CA CA002197662A patent/CA2197662C/en not_active Expired - Fee Related
- 1994-08-15 ES ES94925843T patent/ES2313716T3/en not_active Expired - Lifetime
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- 1994-08-15 KR KR1019970700990A patent/KR100358448B1/en not_active IP Right Cessation
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ATE412887T1 (en) | 2008-11-15 |
DE69435159D1 (en) | 2008-12-11 |
KR970705019A (en) | 1997-09-06 |
JPH10504105A (en) | 1998-04-14 |
AU706107B2 (en) | 1999-06-10 |
ES2313716T3 (en) | 2009-03-01 |
CA2197662C (en) | 2007-07-10 |
HK1001415A1 (en) | 1998-06-19 |
EP0783681A1 (en) | 1997-07-16 |
KR100358448B1 (en) | 2003-05-09 |
CA2197662A1 (en) | 1996-02-22 |
EP0783681A4 (en) | 1998-11-25 |
AU7562694A (en) | 1996-03-07 |
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